Methods and materials for detecting genetic or epigenetic elements

ABSTRACT

This document provides methods and materials for detecting genetic and/or epigenetic elements. For example, methods and materials for detecting the presence or absence of target nucleic acid containing a genetic or epigenetic element, methods and materials for detecting the amount of target nucleic acid containing a genetic or epigenetic element within a sample, kits for detecting the presence or absence of target nucleic acid containing a genetic or epigenetic element, kits for detecting the amount of target nucleic acid containing a genetic or epigenetic element present within a sample, and methods for making such kits are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 61/304,793, filed Feb. 15, 2010. The disclosure ofthe prior application is considered part of (and is incorporated byreference in) the disclosure of this application.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in detectinggenetic and/or epigenetic elements. For example, this document relatesto methods and materials involved in using an enzymatic amplificationcascade of restriction endonucleases to detect genetic and/or epigeneticelements present within an organism (e.g., a human).

2. Background

Many aspects of an organism's phenotype are controlled by the genotypeof that organism. In other words, the genetic makeup of an organism cancontrol the traits of that organism. Thus, the presence or absence ofcertain genetic elements such as single nucleotide polymorphisms (SNPs),sequence deletions, or sequence additions present within an organism'sgenome can provide important information about the organism's healthand/or susceptibilities to certain diseases or disorders. Likewise,epigenetic elements such as methylated DNA can control or influence anorganism's phenotype. Thus, the presence or absence of certainepigenetic elements such as methylated DNA present within an organismcan provide important information about the organism's health and/orsusceptibilities to certain diseases or disorders.

SUMMARY

This document provides methods and materials for detecting geneticand/or epigenetic elements. For example, this document relates tomethods and materials involved in using an enzymatic amplificationcascade of restriction endonucleases to detect genetic and/or epigeneticelements present within an organism (e.g., a human). Information aboutan organism's genotype can be important for understanding thatorganism's health and/or susceptibilities to certain diseases ordisorders. For example, the presence of certain genetic elements in ahuman's genome (e.g., genetic markers such as single nucleotidepolymorphisms (SNPs), sequence deletions, or sequence additions) canindicate that that particular human has an enzyme deficiency or issusceptible to developing a certain disease. Likewise, information aboutepigenetic elements that may influence the phenotype of an organism canbe important for understanding that organism's health and/orsusceptibilities to certain diseases or disorders. For example, thepresence of certain epigenetic elements such as methylated DNA canindicate that a human has a particular type of cancer.

In some cases, this document provides methods and materials fordetecting target nucleic acid that contains a genetic element orepigenetic element. For example, this document provides methods andmaterials for detecting the presence or absence of target nucleic acid(e.g., target nucleic acid containing a particular genetic element) inan organism's genome, methods and materials for detecting the presenceor absence of target nucleic acid that contains an epigenetic element(e.g., methylated DNA) in a cell of an organism, kits for detecting thepresence or absence of target nucleic acid (e.g., target nucleic acidcontaining a particular genetic element) in an organism's genome, kitsfor detecting the presence or absence of target nucleic acid thatcontains an epigenetic element (e.g., methylated DNA) in a cell of anorganism, and methods for making such kits.

In general, the methods and materials provided herein can includeperforming an enzymatic amplification cascade of restrictionendonucleases as described herein to detect target nucleic acidindicative of a genetic and/or epigenetic element in a manner that israpid, inexpensive, sensitive, and specific. For example, a sample(e.g., a sample of genomic nucleic acid or a sample of nucleic acid froma cell or tissue) can be obtained from an organism (e.g., a human)and/or processed such that target nucleic acid, if present within thesample, is capable of hybridizing to probe nucleic acid of an enzymaticamplification cascade of restriction endonucleases described herein. Insome cases, such an obtained and/or processed sample can be assessed forthe presence, absence, or amount of target nucleic acid using anenzymatic amplification cascade of restriction endonucleases describedherein without using a nucleic acid amplification technique (e.g., aPCR-based nucleic acid technique). Assessing samples (e.g., biologicalsamples) for the presence, absence, or amount of target nucleic acidusing an enzymatic amplification cascade of restriction endonucleasesdescribed herein without using a nucleic acid amplification techniquecan allow patients as well as medical, laboratory, or veterinarianpersonnel (e.g., clinicians, physicians, physician's assistants,laboratory technicians, research scientists, and veterinarians) to testorganisms for possible genetic and/or epigenetic elements using anucleic acid-based assay without the need for potentially expensivethermal cycling devices and potentially time consuming thermal cyclingtechniques. In addition, the methods and materials provided herein canallow patients as well as medical, laboratory, or veterinarian personnelto detect any type of genetic and/or epigenetic element suspected ofbeing present within an organism (e.g., a mammal such as a human). Forexample, the methods and materials provided herein can be used to detectthe presence or absence of a single nucleotide polymorphism within thegenome of a human.

In general, one aspect of this document features a method for assessingan organism for a genetic or epigenetic element. The method comprises,or consists essentially of, (a) contacting a sample from the organismwith a probe nucleic acid comprising an amplifying restrictionendonuclease and a nucleotide sequence complementary to a sequence of atarget nucleic acid containing the genetic or epigenetic element underconditions wherein, if the target nucleic acid is present in the sample,at least a portion of the target nucleic acid hybridizes to at least aportion of the probe nucleic acid to form a double-stranded portion ofnucleic acid comprising a restriction endonuclease cut site, (b)contacting the double-stranded portion of nucleic acid with arecognition restriction endonuclease having the ability to cut thedouble-stranded portion of nucleic acid at the restriction endonucleasecut site under conditions wherein the recognition restrictionendonuclease cleaves the double-stranded portion of nucleic acid at therestriction endonuclease cut site, thereby separating a portion of theprobe nucleic acid comprising the amplifying restriction endonucleasefrom at least another portion of the probe nucleic acid, (c) contactingthe portion of the probe nucleic acid comprising the amplifyingrestriction endonuclease with a reporter nucleic acid comprising adouble-stranded portion of nucleic acid comprising a restrictionendonuclease cut site of the amplifying restriction endonuclease underconditions wherein the amplifying restriction endonuclease cleaves thereporter nucleic acid at the restriction endonuclease cut site of theamplifying restriction endonuclease, thereby separating a portion of thereporter nucleic acid from at least another portion of the reporternucleic acid, and (d) determining the presence or absence of the portionof the reporter nucleic acid, wherein the presence of the portion of thereporter nucleic acid indicates that the sample contains the targetnucleic acid, thereby indicating that the organism contains the geneticor epigenetic element, and wherein the absence of the portion of thereporter nucleic acid indicates that the sample does not contain thetarget nucleic acid, thereby indicating that the organism does notcontain the genetic or epigenetic element. The organism can be a human.The organism can be a mammal. The mammal can be selected from the groupconsisting of bovine, porcine, and equine species. The organism can be aplant. The plant can be selected from the group consisting of trees,flowers, shrubs, grains, grasses, and legumes. The method comprisesassessing the organism for the genetic element. The genetic element canbe an allelic variant known to exist in the species of the organism. Thegenetic element can be a single nucleotide polymorphism. The method cancomprise assessing the organism for the epigenetic element. Theepigenetic element can be a methylated DNA sequence. The sample can beselected from the group consisting of blood samples, hair samples, skinsamples, throat swab samples, cheek swab samples, tissue samples,cellular samples, and tumor samples. Prior to step (a), the sample canbe a sample that was processed to remove non-nucleic acid material fromthe sample, thereby increasing the concentration of nucleic acid, ifpresent, within the sample. The sample can be a sample that wassubjected to a nucleic acid extraction technique. Prior to step (a), thesample can be a sample that was subjected to a nucleic acidamplification technique to increase the concentration of one or morenucleic acids, if present, within the sample. The sample can be a samplethat was subjected to a PCR-based technique designed to amplify thetarget nucleic acid. Prior to step (a), the method can comprise removingnon-nucleic acid material from the sample, thereby increasing theconcentration of nucleic acid, if present, within the sample. Theremoving can comprise performing a nucleic acid extraction technique.Prior to step (a), the method can comprise performing a nucleic acidamplification technique to increase the concentration of one or morenucleic acids, if present, within the sample. The nucleic acidamplification technique can comprise a PCR-based technique designed toamplify the target nucleic acid. Prior to step (a), the method cancomprise removing non-nucleic acid material from the sample, therebyincreasing the concentration of nucleic acid, if present, within thesample, and performing a nucleic acid amplification technique toincrease the concentration of one or more nucleic acids, if present,within the sample. The probe nucleic acid can be single-stranded probenucleic acid. The probe nucleic acid can be attached to a solid support.The probe nucleic acid can be directly attached to a solid support. Theportion of the probe nucleic acid comprising the amplifying restrictionendonuclease can be released from the solid support via the step (b).Step (a) and step (b) can be performed in the same compartment, or step(a), step (b), and step (c) can be performed in the same compartment, orstep (a), step (b), step (c), and step (d) can be performed in the samecompartment. Step (a) and step (b) can be performed in a firstcompartment, and step (c) can be performed in a second compartment. Step(a) and step (b) can be performed by adding the sample to a compartmentcomprising the probe nucleic acid and the recognition restrictionendonuclease. The probe nucleic acid can comprise (i) a single-strandedportion comprising the nucleotide sequence complementary to the sequenceof the target nucleic acid and (ii) a double-stranded portion. The probenucleic acid can comprise a first nucleic acid strand comprising thenucleotide sequence complementary to the sequence of the target nucleicacid hybridized to a second nucleic acid strand comprising theamplifying restriction endonuclease. The first nucleic acid strand canbe attached to a solid support. The first nucleic acid strand can bedirectly attached to a solid support. A portion of the second nucleicacid strand can hybridize with the first nucleic acid strand to form thedouble-stranded portion. The portion of the probe nucleic acidcomprising the amplifying restriction endonuclease that is separatedfrom the at least another portion of the probe nucleic acid in step (b)can comprise a portion of the first nucleic acid strand and all of thesecond strand. The portion of the probe nucleic acid comprising theamplifying restriction endonuclease that is separated from the at leastanother portion of the probe nucleic acid in step (b) can comprise atleast a portion of the target nucleic acid.

In some cases, the method can comprise using a plurality of the probenucleic acid in the step (a). The method can comprise using a pluralityof the reporter nucleic acid in the step (c). The reporter nucleic acidin the step (c) can be in molar excess of the portion of the probenucleic acid comprising the amplifying restriction endonuclease from thestep (b). The number of molecules of the portion of the probe nucleicacid comprising the amplifying restriction endonuclease that isseparated from the at least another portion of the probe nucleic acid instep (b) can be in an essentially linear relationship to the number ofmolecules of the target nucleic acid present in the sample. The reporternucleic acid can be attached to a solid support. The reporter nucleicacid can be directly attached to a solid support. The reporter nucleicacid can comprise a single-stranded portion of nucleic acid. Thereporter nucleic acid can comprise a label. The label can be afluorescent label, a radioactive label, an enzyme label, or a redoxlabel. The portion of the reporter nucleic acid that is separated fromthe at least another portion of the reporter nucleic acid can comprisethe label. The reporter nucleic acid can comprise a first nucleic acidstrand comprising the label hybridized to a second nucleic acid strand.The second nucleic acid strand can be attached to a solid support. Thesecond nucleic acid strand can be directly attached to a solid support.A portion of the first nucleic acid strand can hybridize with the secondnucleic acid strand to form the double-stranded portion of nucleic acidcomprising the restriction endonuclease cut site of the amplifyingrestriction endonuclease. The reporter nucleic acid can comprise a thirdnucleic acid strand. The third nucleic acid strand can hybridize withthe second nucleic acid strand to form the double-stranded portion ofnucleic acid comprising the restriction endonuclease cut site of theamplifying restriction endonuclease. The reporter nucleic acid can beattached to a solid support, and the portion of the reporter nucleicacid that is separated from the at least another portion of the reporternucleic acid and that comprises the label can be released from the solidsupport via the step (c). The determining step (d) can comprisedetecting the label. The label can be a fluorescent label, and thedetermining step (d) comprises detecting the fluorescent label. Thedetermining step (d) can comprise detecting the portion of the reporternucleic acid separated from the at least another portion of the reporternucleic acid using a capillary electrophoresis technique. The steps (a),(b), and (c) can be performed without nucleic acid amplification, or thesteps (a), (b), (c), and (d) can be performed without nucleic acidamplification. The determining step can comprise determining the amountof the target nucleic acid present within the sample.

In another aspect, this document features a method for an organism for agenetic or epigenetic element. The method comprises, or consistsessentially of, (a) contacting a sample from the mammal with a probenucleic acid comprising an initial amplifying restriction endonucleaseand a nucleotide sequence complementary to a sequence of a targetnucleic acid containing the genetic or epigenetic element underconditions wherein, if the target nucleic acid is present in the sample,at least a portion of the target nucleic acid hybridizes to at least aportion of the probe nucleic acid to form a double-stranded portion ofnucleic acid comprising a restriction endonuclease cut site, (b)contacting the double-stranded portion of nucleic acid with arecognition restriction endonuclease having the ability to cut thedouble-stranded portion of nucleic acid at the restriction endonucleasecut site under conditions wherein the recognition restrictionendonuclease cleaves the double-stranded portion of nucleic acid at therestriction endonuclease cut site, thereby separating a portion of theprobe nucleic acid comprising the initial amplifying restrictionendonuclease from at least another portion of the probe nucleic acid,(c) contacting the portion of the probe nucleic acid comprising theinitial amplifying restriction endonuclease with a first nucleic acidcomprising a secondary amplifying restriction endonuclease and adouble-stranded portion of nucleic acid comprising a restrictionendonuclease cut site of the initial amplifying restriction endonucleaseunder conditions wherein the initial amplifying restriction endonucleasecleaves the first nucleic acid at the restriction endonuclease cut siteof the initial amplifying restriction endonuclease, thereby separating aportion of the first nucleic acid comprising the secondary amplifyingrestriction endonuclease from at least another portion of the firstnucleic acid, (d) contacting the portion of the first nucleic acidcomprising the secondary amplifying restriction endonuclease with asecond nucleic acid comprising the initial amplifying restrictionendonuclease and a double-stranded portion of nucleic acid comprising arestriction endonuclease cut site of the secondary amplifyingrestriction endonuclease under conditions wherein the secondaryamplifying restriction endonuclease cleaves the second nucleic acid atthe restriction endonuclease cut site of the secondary amplifyingrestriction endonuclease, thereby separating a portion of the secondnucleic acid comprising the initial amplifying restriction endonucleasefrom at least another portion of the second nucleic acid, (e) contactingthe portion of the second nucleic acid comprising the initial amplifyingrestriction endonuclease with a reporter nucleic acid comprising adouble-stranded portion of nucleic acid comprising a restrictionendonuclease cut site of the initial amplifying restriction endonucleaseunder conditions wherein the initial amplifying restriction endonucleasecleaves the reporter nucleic acid at the restriction endonuclease cutsite of the initial amplifying restriction endonuclease, therebyseparating a portion of the reporter nucleic acid from at least anotherportion of the reporter nucleic acid, and (f) determining the presenceor absence of the portion of the reporter nucleic acid, wherein thepresence of the portion of the reporter nucleic acid indicates that thesample contains the target nucleic acid, thereby indicating that theorganism contains the genetic or epigenetic element, and wherein theabsence of the portion of the reporter nucleic acid indicates that thesample does not contain the target nucleic acid, thereby indicating thatthe organism does not contain the genetic or epigenetic element. Theorganism can be a human. The organism can be a mammal. The mammal can beselected from the group consisting of bovine, porcine, and equinespecies. The organism can be a plant. The plant can be selected from thegroup consisting of trees, flowers, shrubs, grains, grasses, andlegumes. The method can comprise assessing the organism for the geneticelement. The genetic element can be an allelic variant known to exist inthe species of the organism. The genetic element can be a singlenucleotide polymorphism. The method can comprise assessing the organismfor the epigenetic element. The epigenetic element can be a methylatedDNA sequence. The sample can be selected from the group consisting ofblood samples, hair samples, skin samples, throat swab samples, cheekswab samples, tissue samples, cellular samples, and tumor samples. Priorto step (a), the sample can be a sample that was processed to removenon-nucleic acid material from the sample, thereby increasing theconcentration of nucleic acid, if present, within the sample. The samplecan be a sample that was subjected to a nucleic acid extractiontechnique. Prior to step (a), the sample can be a sample that wassubjected to a nucleic acid amplification technique to increase theconcentration of one or more nucleic acids, if present, within thesample. The sample can be a sample that was subjected to a PCR-basedtechnique designed to amplify the target nucleic acid. Prior to step(a), the method can comprise removing non-nucleic acid material from thesample, thereby increasing the concentration of nucleic acid, ifpresent, within the sample. The removing can comprise performing anucleic acid extraction technique. Prior to step (a), the method cancomprise performing a nucleic acid amplification technique to increasethe concentration of one or more nucleic acids, if present, within thesample. The nucleic acid amplification technique can comprise aPCR-based technique designed to amplify the target nucleic acid. Priorto step (a), the method can comprise removing non-nucleic acid materialfrom the sample, thereby increasing the concentration of nucleic acid,if present, within the sample, and performing a nucleic acidamplification technique to increase the concentration of one or morenucleic acids, if present, within the sample. The probe nucleic acid canbe single-stranded probe nucleic acid. The probe nucleic acid can beattached to a solid support. The probe nucleic acid can be directlyattached to a solid support. The portion of the probe nucleic acidcomprising the initial amplifying restriction endonuclease can bereleased from the solid support via the step (b). Step (a) and step (b)can be performed in the same compartment, step (a), step (b), and step(c) can be performed in the same compartment, step (a), step (b), step(c), and step (d) can be performed in the same compartment, step (a),step (b), step (c), step (d), and step (e) can be performed in the samecompartment, or step (a), step (b), step (c), step (d), step (e), andstep (f) can be performed in the same compartment. Step (c) and step (d)can be performed in the same compartment. Step (a) and step (b) can beperformed in a first compartment, and step (c) and step (d) can beperformed in a second compartment. Step (a) and step (b) can beperformed by adding the sample to a compartment comprising the probenucleic acid and the recognition restriction endonuclease. Step (c) andstep (d) can be performed by adding the portion of the probe nucleicacid comprising the initial amplifying restriction endonuclease to acompartment comprising the first nucleic acid and the second nucleicacid. The probe nucleic acid can comprise (i) a single-stranded portioncomprising the nucleotide sequence complementary to the sequence of thetarget nucleic acid and (ii) a double-stranded portion. The probenucleic acid can comprise a first nucleic acid strand comprising thenucleotide sequence complementary to the sequence of the target nucleicacid hybridized to a second nucleic acid strand comprising the initialamplifying restriction endonuclease. The first nucleic acid strand canbe attached to a solid support. The first nucleic acid strand can bedirectly attached to a solid support. A portion of the second nucleicacid strand can hybridize with the first nucleic acid strand to form thedouble-stranded portion. The portion of the probe nucleic acidcomprising the initial amplifying restriction endonuclease that isseparated from the at least another portion of the probe nucleic acid instep (b) can comprise a portion of the first nucleic acid strand and allof the second strand. The portion of the probe nucleic acid comprisingthe initial amplifying restriction endonuclease that is separated fromthe at least another portion of the probe nucleic acid in step (b) cancomprise at least a portion of the target nucleic acid.

In some cases, the method can comprise using a plurality of the probenucleic acid in the step (a). The method can comprise using a pluralityof the reporter nucleic acid in the step (e). The reporter nucleic acidin the step (e) can be in molar excess of the portion of the probenucleic acid comprising the initial amplifying restriction endonucleasefrom the step (b). The number of molecules of the portion of the probenucleic acid comprising the initial amplifying restriction endonucleasethat is separated from the at least another portion of the probe nucleicacid in step (b) can be in an essentially linear relationship to thenumber of molecules of the target nucleic acid present in the sample.The first nucleic acid and the second nucleic acid can be attached to asolid support. The first nucleic acid and the second nucleic acid can bedirectly attached to a solid support. The first nucleic acid and thesecond nucleic acid can be attached to a solid support in the samecompartment. The portion of the first nucleic acid comprising thesecondary amplifying restriction endonuclease can be released from thesolid support via the step (c). The portion of the second nucleic acidcomprising the initial amplifying restriction endonuclease can bereleased from the solid support via the step (d). The first nucleic acidcan comprise a first nucleic acid strand comprising the secondaryamplifying restriction endonuclease hybridized to a second nucleic acidstrand to form the double-stranded portion of nucleic acid comprisingthe restriction endonuclease cut site of the initial amplifyingrestriction endonuclease. The first nucleic acid strand can be attachedto a solid support. The first nucleic acid strand can be directlyattached to a solid support. The second nucleic acid strand can beattached to a solid support. The second nucleic acid strand can bedirectly attached to a solid support. The second nucleic acid cancomprise a first nucleic acid strand comprising the initial amplifyingrestriction endonuclease hybridized to a second nucleic acid strand toform the double-stranded portion of nucleic acid comprising therestriction endonuclease cut site of the secondary amplifyingrestriction endonuclease. The first nucleic acid strand can be attachedto a solid support. The first nucleic acid strand can be directlyattached to a solid support. The second nucleic acid strand can beattached to a solid support. The second nucleic acid strand can bedirectly attached to a solid support. The reporter nucleic acid can beattached to a solid support. The reporter nucleic acid can be directlyattached to a solid support. The reporter nucleic acid can comprise asingle-stranded portion of nucleic acid. The reporter nucleic acid cancomprise a label. The label can be a fluorescent label, a radioactivelabel, an enzyme label, or a redox label. The portion of the reporternucleic acid that is separated from the at least another portion of thereporter nucleic acid can comprise the label. The reporter nucleic acidcan comprise a first nucleic acid strand comprising the label hybridizedto a second nucleic acid strand. The second nucleic acid strand can beattached to a solid support. The second nucleic acid strand can bedirectly attached to a solid support. A portion of the first nucleicacid strand can hybridize with the second nucleic acid strand to formthe double-stranded portion of nucleic acid comprising the restrictionendonuclease cut site of the initial amplifying restrictionendonuclease. The reporter nucleic acid can comprise a third nucleicacid strand. The third nucleic acid strand can hybridize with the secondnucleic acid strand to form the double-stranded portion of nucleic acidcomprising the restriction endonuclease cut site of the initialamplifying restriction endonuclease. The reporter nucleic acid can beattached to a solid support, and the portion of the reporter nucleicacid that is separated from the at least another portion of the reporternucleic acid and that comprises the label can be released from the solidsupport via the step (e). The determining step (f) can comprisedetecting the label. The label can be a fluorescent label, and thedetermining step (f) can comprise detecting the fluorescent label. Thedetermining step (f) can comprise detecting the portion of the reporternucleic acid separated from the at least another portion of the reporternucleic acid using a capillary electrophoresis technique. Steps (a),(b), (c), (d), and (e) can be performed without nucleic acidamplification, or steps (a), (b), (c), (d), (e), and (f) can beperformed without nucleic acid amplification. The determining step cancomprise determining the amount of the target nucleic acid presentwithin the sample.

In another aspect, this document features a method for assessing anorganism for a genetic or epigenetic element. The method comprises, orconsists essentially of, (a) contacting a sample from the mammal with aprobe nucleic acid comprising an initial amplifying restrictionendonuclease and a nucleotide sequence complementary to a sequence of atarget nucleic acid containing the genetic or epigenetic element underconditions wherein, if the target nucleic acid is present in the sample,at least a portion of the target nucleic acid hybridizes to at least aportion of the probe nucleic acid to form a double-stranded portion ofnucleic acid comprising a restriction endonuclease cut site, (b)contacting the double-stranded portion of nucleic acid with arecognition restriction endonuclease having the ability to cut thedouble-stranded portion of nucleic acid at the restriction endonucleasecut site under conditions wherein the recognition restrictionendonuclease cleaves the double-stranded portion of nucleic acid at therestriction endonuclease cut site, thereby separating a portion of theprobe nucleic acid comprising the initial amplifying restrictionendonuclease from at least another portion of the probe nucleic acid,(c) contacting the portion of the probe nucleic acid comprising theinitial amplifying restriction endonuclease with a first reporternucleic acid comprising a secondary amplifying restriction endonucleaseand a double-stranded portion of nucleic acid comprising a restrictionendonuclease cut site of the initial amplifying restriction endonucleaseunder conditions wherein the initial amplifying restriction endonucleasecleaves the first reporter nucleic acid at the restriction endonucleasecut site of the initial amplifying restriction endonuclease, therebyseparating a portion of the first nucleic acid comprising the secondaryamplifying restriction endonuclease from at least another portion of thefirst nucleic acid, (d) contacting the portion of the first reporternucleic acid comprising the secondary amplifying restrictionendonuclease with a second reporter nucleic acid comprising the initialamplifying restriction endonuclease and a double-stranded portion ofnucleic acid comprising a restriction endonuclease cut site of thesecondary amplifying restriction endonuclease under conditions whereinthe initial amplifying restriction endonuclease cleaves the secondnucleic acid at the restriction endonuclease cut site of the secondaryamplifying restriction endonuclease, thereby separating a portion of thesecond nucleic acid comprising the initial amplifying restrictionendonuclease from at least another portion of the second nucleic acid,and (e) determining the presence or absence of the portion of the firstreporter nucleic acid, the second reporter nucleic acid, or both thefirst reporter nucleic acid and the second reporter nucleic acid,wherein the presence indicates that the sample contains the targetnucleic acid, thereby indicating that the organism contains the geneticor epigenetic element, and wherein the absence indicates that the sampledoes not contain the target nucleic acid, thereby indicating that theorganism does not contain the genetic or epigenetic element. Theorganism can be a human. The organism can be a mammal. The mammal can beselected from the group consisting of bovine, porcine, and equinespecies. The organism can be a plant. The plant can be selected from thegroup consisting of trees, flowers, shrubs, grains, grasses, andlegumes. The method can comprise assessing the organism for the geneticelement. The genetic element can be an allelic variant known to exist inthe species of the organism. The genetic element can be a singlenucleotide polymorphism. The method can comprise assessing the organismfor the epigenetic element. The epigenetic element can be a methylatedDNA sequence. The sample can be selected from the group consisting ofblood samples, hair samples, skin samples, throat swab samples, cheekswab samples, tissue samples, cellular samples, and tumor samples. Priorto step (a), the sample can be a sample that was processed to removenon-nucleic acid material from the sample, thereby increasing theconcentration of nucleic acid, if present, within the sample. The samplecan be a sample that was subjected to a nucleic acid extractiontechnique. Prior to step (a), the sample can be a sample that wassubjected to a nucleic acid amplification technique to increase theconcentration of one or more nucleic acids, if present, within thesample. The sample can be a sample that was subjected to a PCR-basedtechnique designed to amplify the target nucleic acid. Prior to step(a), the method can comprise removing non-nucleic acid material from thesample, thereby increasing the concentration of nucleic acid, ifpresent, within the sample. The removing can comprise performing anucleic acid extraction technique. Prior to step (a), the method cancomprise performing a nucleic acid amplification technique to increasethe concentration of one or more nucleic acids, if present, within thesample. The nucleic acid amplification technique can comprise aPCR-based technique designed to amplify the target nucleic acid. Priorto step (a), the method can comprise removing non-nucleic acid materialfrom the sample, thereby increasing the concentration of nucleic acid,if present, within the sample, and performing a nucleic acidamplification technique to increase the concentration of one or morenucleic acids, if present, within the sample. The probe nucleic acid canbe single-stranded probe nucleic acid. The probe nucleic acid can beattached to a solid support. The probe nucleic acid can be directlyattached to a solid support. The portion of the probe nucleic acidcomprising the initial amplifying restriction endonuclease can bereleased from the solid support via the step (b). Step (a) and step (b)can be performed in the same compartment, step (a), step (b), and step(c) can be performed in the same compartment, step (a), step (b), step(c), and step (d) can be performed in the same compartment, or step (a),step (b), step (c), step (d), and step (e) can be performed in the samecompartment. Step (c) and step (d) can be performed in the samecompartment. Step (a) and step (b) can be performed in a firstcompartment, and step (c) and step (d) can be performed in a secondcompartment. Step (a) and step (b) can be performed by adding the sampleto a compartment comprising the probe nucleic acid and the recognitionrestriction endonuclease. Step (c) and step (d) can be performed byadding the portion of the probe nucleic acid comprising the initialamplifying restriction endonuclease to a compartment comprising thefirst reporter nucleic acid and the second reporter nucleic acid. Theprobe nucleic acid can comprise (i) a single-stranded portion comprisingthe nucleotide sequence complementary to the sequence of the targetnucleic acid and (ii) a double-stranded portion. The probe nucleic acidcan comprise a first nucleic acid strand comprising the nucleotidesequence complementary to the sequence of the target nucleic acidhybridized to a second nucleic acid strand comprising the initialamplifying restriction endonuclease. The first nucleic acid strand canbe attached to a solid support. The first nucleic acid strand can bedirectly attached to a solid support. A portion of the second nucleicacid strand can hybridize with the first nucleic acid strand to form thedouble-stranded portion. The portion of the probe nucleic acidcomprising the initial amplifying restriction endonuclease that isseparated from the at least another portion of the probe nucleic acid instep (b) can comprise a portion of the first nucleic acid strand and allof the second strand. The portion of the probe nucleic acid comprisingthe initial amplifying restriction endonuclease that is separated fromthe at least another portion of the probe nucleic acid in step (b) cancomprise at least a portion of the target nucleic acid.

In some cases, the method can comprise using a plurality of the probenucleic acid in the step (a). The method can comprise using a pluralityof the first reporter nucleic acid in the step (c). The first reporternucleic acid in the step (c) can be in molar excess of the portion ofthe probe nucleic acid comprising the initial amplifying restrictionendonuclease from the step (b). The method can comprise using aplurality of the second reporter nucleic acid in the step (d). Thesecond reporter nucleic acid in the step (d) can be in molar excess ofthe portion of the probe nucleic acid comprising the initial amplifyingrestriction endonuclease from the step (b). The number of molecules ofthe portion of the probe nucleic acid comprising the initial amplifyingrestriction endonuclease that is separated from the at least anotherportion of the probe nucleic acid in step (b) can be in an essentiallylinear relationship to the number of molecules of the target nucleicacid present in the sample. The first reporter nucleic acid and thesecond reporter nucleic acid can be attached to a solid support. Thefirst reporter nucleic acid and the second reporter nucleic acid can bedirectly attached to a solid support. The first reporter nucleic acidand the second reporter nucleic acid can be attached to a solid supportin the same compartment. The portion of the first reporter nucleic acidcomprising the secondary amplifying restriction endonuclease can bereleased from the solid support via the step (c). The portion of thesecond reporter nucleic acid comprising the initial amplifyingrestriction endonuclease can be released from the solid support via thestep (d). The first reporter nucleic acid can comprise a label. Thelabel can be a fluorescent label, a radioactive label, an enzyme label,or a redox label. The second reporter nucleic acid can comprise a label.The label can be a fluorescent label, a radioactive label, an enzymelabel, or a redox label. The first reporter nucleic acid and the secondreporter nucleic acid can comprise a label. The first reporter nucleicacid and the second reporter nucleic acid can comprise the same label.The label can be a fluorescent label, a radioactive label, an enzymelabel, or a redox label. The first reporter nucleic acid can be attachedto a solid support, the portion of the first reporter nucleic acid thatis separated from the at least another portion of the first reporternucleic acid can comprise a label, and the portion of the first reporternucleic acid that is separated from the at least another portion of thefirst reporter nucleic acid and that comprises the label can be releasedfrom the solid support via the step (c). The first reporter nucleic acidcan comprise a first nucleic acid strand comprising the secondaryamplifying restriction endonuclease hybridized to a second nucleic acidstrand to form the double-stranded portion of nucleic acid comprisingthe restriction endonuclease cut site of the initial amplifyingrestriction endonuclease. The first nucleic acid strand can be attachedto a solid support. The first nucleic acid strand can be directlyattached to a solid support. The second nucleic acid strand can beattached to a solid support. The second nucleic acid strand can bedirectly attached to a solid support. The first nucleic acid strand cancomprise a label. The label can be a fluorescent label, a radioactivelabel, an enzyme label, or a redox label. The second nucleic acid strandcan comprise a label. The label can be a fluorescent label, aradioactive label, an enzyme label, or a redox label. The secondreporter nucleic acid can be attached to a solid support, the portion ofthe second reporter nucleic acid that is separated from the at leastanother portion of the second reporter nucleic acid can comprise alabel, and the portion of the second reporter nucleic acid that isseparated from the at least another portion of the second reporternucleic acid and that comprises the label can be released from the solidsupport via the step (d). The second reporter nucleic acid can comprisea first nucleic acid strand comprising the initial amplifyingrestriction endonuclease hybridized to a second nucleic acid strand toform the double-stranded portion of nucleic acid comprising therestriction endonuclease cut site of the secondary amplifyingrestriction endonuclease. The first nucleic acid strand can be attachedto a solid support. The first nucleic acid strand can be directlyattached to a solid support. The second nucleic acid strand can beattached to a solid support. The second nucleic acid strand can bedirectly attached to a solid support. The first nucleic acid strand cancomprise a label. The label can be a fluorescent label, a radioactivelabel, an enzyme label, or a redox label. The second nucleic acid strandcan comprise a label. The label can be a fluorescent label, aradioactive label, an enzyme label, or a redox label. The portion of thefirst reporter nucleic acid separated from the at least another portionof the first reporter nucleic acid can comprise a fluorescent label, theportion of the second reporter nucleic acid separated from the at leastanother portion of the second reporter nucleic acid can comprise afluorescent label, and the determining step (e) can comprise detectingthe fluorescent label. The determining step (e) can comprise detectingthe portion of the first reporter nucleic acid separated from the atleast another portion of the first reporter nucleic acid using acapillary electrophoresis technique. The determining step (e) cancomprise detecting the portion of the second reporter nucleic acidseparated from the at least another portion of the second reporternucleic acid using a capillary electrophoresis technique. Steps (a),(b), (c), and (d) can be performed without nucleic acid amplification,or steps (a), (b), (c), (d), and (e) can be performed without nucleicacid amplification. The determining step can comprise determining theamount of the target nucleic acid present within the sample.

In another aspect, this document features a kit for assessing anorganism for a genetic or epigenetic element. The kit comprises, orconsists essentially of, a probe nucleic acid comprising an amplifyingrestriction endonuclease and a nucleotide sequence complementary to asequence of a target nucleic acid containing the genetic or epigeneticelement, wherein at least a portion of the target nucleic acid iscapable of hybridizing to at least a portion of the probe nucleic acidto form a double-stranded portion of nucleic acid comprising arestriction endonuclease cut site. The probe nucleic acid can besingle-stranded probe nucleic acid. The kit can comprise a solidsupport, and the probe nucleic acid can be attached to the solidsupport. A portion of the probe nucleic acid comprising the amplifyingrestriction endonuclease can be releasable from the solid support viacleavage with a recognition restriction endonuclease having the abilityto cleave at the restriction endonuclease cut site. The kit can furthercomprise the recognition restriction endonuclease. The probe nucleicacid can comprise (i) a single-stranded portion comprising thenucleotide sequence complementary to the sequence of the target nucleicacid and (ii) a double-stranded portion. The probe nucleic acid cancomprise a first nucleic acid strand comprising the nucleotide sequencecomplementary to the sequence of the target nucleic acid hybridized to asecond nucleic acid strand comprising the amplifying restrictionendonuclease. The kit can further comprise a reporter nucleic acidcomprising a double-stranded portion of nucleic acid comprising arestriction endonuclease cut site of the amplifying restrictionendonuclease. The kit can comprise a solid support, and the reporternucleic acid can be attached to the solid support. The reporter nucleicacid can be directly attached to the solid support. The reporter nucleicacid can comprise a single-stranded portion of nucleic acid. Thereporter nucleic acid can comprise a label. The label can be afluorescent label, a radioactive label, an enzyme label, or a redoxlabel. A portion of the reporter nucleic acid comprising the label canbe capable of being separated from at least another portion of thereporter nucleic acid via cleavage by the amplifying restrictionendonuclease. The reporter nucleic acid can comprise a first nucleicacid strand comprising the label hybridized to a second nucleic acidstrand. The kit can further comprise: (a) a first signal expansionnucleic acid comprising a secondary amplifying restriction endonucleaseand a double-stranded section having a restriction endonuclease cut sitefor the amplifying restriction endonuclease, and (b) a second signalexpansion nucleic acid comprising the amplifying restrictionendonuclease and a double-stranded section having a restrictionendonuclease cut site for the secondary amplifying restrictionendonuclease. The probe nucleic acid can be lyophilized. All theingredients of the kit can be lyophilized or dry.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other references (e.g.,GenBank® records) mentioned herein are incorporated by reference intheir entirety. In case of conflict, the present specification,including definitions, will control. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting an exemplary method for detecting targetnucleic acid using probe nucleic acid, a recognition restrictionendonuclease, and reporter nucleic acid.

FIG. 2 is a schematic of an exemplary configuration of probe nucleicacid that can be used with the methods and materials provided herein fordetecting target nucleic acid.

FIG. 3 is a schematic depicting an exemplary method for detecting targetnucleic acid using probe nucleic acid, a recognition restrictionendonuclease, first signal expansion nucleic acid, second signalexpansion nucleic acid, and reporter nucleic acid.

FIG. 4 is a schematic of an exemplary configuration of first signalexpansion nucleic acid and second signal expansion nucleic acid that canbe used with the methods and materials provided herein for detectingtarget nucleic acid. Such first signal expansion nucleic acid and secondsignal expansion nucleic acid can be used with or without reporternucleic acid. When used without a separate reporter nucleic acid step,such signal expansion nucleic acid can be referred to as reporternucleic acid.

FIG. 5 is a schematic of an exemplary configuration of first signalexpansion nucleic acid and second signal expansion nucleic acid that canbe used with the methods and materials provided herein for detectingtarget nucleic acid. Such first signal expansion nucleic acid and secondsignal expansion nucleic acid can be used with or without reporternucleic acid. When used without a separate reporter nucleic acid step,such signal expansion nucleic acid can be referred to as reporternucleic acid.

FIG. 6 contains line graphs demonstrating the effect of targetoligonucleotide concentration (A) and recognition restrictionendonuclease concentration (B) on the cleavage of HRP-labeled nucleicacid as detected by the formation of colored reaction product.

FIG. 7 is a schematic of an exemplary configuration for a single-use,pen-style point of care device.

FIG. 8 is a diagram of an example of a method that can be used to detectmethylated target DNA. In this example, target DNA is hybridized withthe same probe in two different compartments (e.g., wells). Then, arecognition restriction endonuclease (Rr1), which ismethylation-sensitive, is added to the compartment 1. Rr1 can onlycleave its corresponding restriction site if it is unmodified bymethylation (unmethylated C). Another recognition restrictionendonuclease (Rr2), which is methylation-insensitive, is added to thecompartment 2. Rr2 can cleave both methylated and unmethylated versionsof the target DNA. Signal detection for both compartments is thencompleted as described herein. The resultant signal from compartment 1corresponds exclusively to unmethylated target, while the resultantsignal from compartment 2 corresponds to all target DNA notwithstandingits methylation state. Thus, the amount of methylated target can becalculated by subtracting the compartment 1 signal from the compartment2 signal. This example uses a pair of methylation sensitive andmethylation insensitive restriction endonucleases that recognize and cutthe same site/sequence.

FIG. 9 is a diagram of an example of a method that can be used to detectmethylated target DNA using two different methylation sensitiverecognition restriction endonucleases (Rr1 and Rr1) that have differentcut sites (cut site A and cut site B).

FIG. 10 is a diagram demonstrating how target nucleic acid thatperfectly matches (perfect match, PM) the probe nucleic acid at thegenerated cut site is cleaved by the recognition restrictionendonuclease, while nucleic acid lacking the perfect match (mismatch,MM) with the probe nucleic acid at the generated cut site is not cleavedby the recognition restriction endonuclease.

FIG. 11 contains a double stranded section of DNA (SEQ ID NO:1) thatcontains the recognition site and cleavage site for a FokI restrictionendonuclease.

FIG. 12 is a diagram of an example of a method that can be used todifferentiate between any target nucleic acid that perfectly matches aportion of probe nucleic acid and any nucleic acid that does notperfectly match that portion of probe nucleic acid. Panel A is a diagramdemonstrating how target nucleic acid that perfectly matches (perfectmatch, PM) the probe nucleic acid generates a cut site, of a recognitionrestriction endonuclease that has separate recognition and cleavagesites (e.g., an FokI restriction endonuclease), and together with theprobe nucleic acid is cleaved by the recognition restrictionendonuclease. Panel B is a diagram demonstrating how nucleic acidlacking a perfect match (e.g., nucleic acid containing a SNP) with theprobe nucleic acid does not generate a cut site, of a recognitionrestriction endonuclease that has separate recognition and cleavagesites (e.g., an FokI restriction endonuclease), and together with theprobe nucleic acid is not cleaved by the recognition restrictionendonuclease.

FIG. 13 depicts two probe nucleic acids, one designed to detect targetnucleic acid of an un-mutated version of an thiopurineS-methyltransferase (TPMT; EC 2.1.1.67) enzyme (A; SEQ ID NO:13 linkedto SEQ ID NO:11), and one designed to detect target nucleic acid of amutated version of TPMT carrying a SNP in the codon 154 (B; SEQ ID NO:13linked to SEQ ID NO:12).

DETAILED DESCRIPTION

This document provides methods and materials for detecting geneticand/or epigenetic elements. For example, this document relates tomethods and materials involved in using an enzymatic amplificationcascade of restriction endonucleases to detect genetic and/or epigeneticelements present within an organism (e.g., a human). In some cases, thisdocument provides methods and materials for detecting target nucleicacid that contains a genetic element or epigenetic element. For example,this document provides methods and materials for detecting the presenceor absence of target nucleic acid (e.g., target nucleic acid containinga particular genetic element) in an organism's genome, methods andmaterials for detecting the presence or absence of target nucleic acidthat contains an epigenetic element (e.g., methylated DNA) in a cell ofan organism, kits for detecting the presence or absence of targetnucleic acid (e.g., target nucleic acid containing a particular geneticelement) in an organism's genome, kits for detecting the presence orabsence of target nucleic acid that contains an epigenetic element(e.g., methylated DNA) in a cell of an organism, and methods for makingsuch kits.

Any type of organism (e.g., plant or animal) can be assessed using themethods and materials provided herein to determine whether or not theorganism contains a genetic and/or epigenetic element. Examples oforganisms that can be assessed using the methods and materials providedherein to determine whether or not the organism contains a geneticand/or epigenetic element include, without limitation, plants (e.g.,trees, flowers, shrubs, grains, grasses, and legumes), mammals (e.g.,humans, dogs, cats, cows, horses, pigs, sheep, goats, monkeys, buffalo,bears, whales, and dolphins), avian species (e.g., chickens, turkeys,ostrich, emus, cranes, and falcons), and non-mammalian animals (e.g.,mollusks, frogs, lizards, snakes, and insects). For example, plant cropssuch as corn, soybeans, wheat, and rice can be assessed for the presenceor absence of genetic elements such as possible introduced transgenes,transposable elements, or polymorphisms.

Any type of biological sample can be used with the methods and materialsprovided herein to assess an organism for a particular genetic orepigenetic element. For example, any type of biological sample that isobtained from an organism to be tested and that contains the organism'snucleic acid (e.g., potentially methylated nucleic acid or genomic DNA)can be used as described herein. Examples of samples that can be used asdescribed herein include, without limitation, blood samples (e.g., 50 mLcollection of a patient's blood), serum samples, hair samples, skinsamples, tissue samples (e.g., tissue biopsy samples), bone marrowsamples, tumor samples, amniotic fluid samples, throat or cheek swabsamples (e.g., a buccal smear sample), and mouthwash samples. In somecases, a sample used herein can be a serum sample prepared from wholeblood such that circulating DNA is present in the sample as describedelsewhere (Sunami et al., Methods Mol. Biol., 507:349-56 (2009)).

The methods and materials provided herein can be used to assess anorganism for any type of genetic or epigenetic element. Examples ofpossible genetic elements that can be assessed using the methods andmaterials provided herein include, without limitation, wild-type orcommon standard allele sequences of an organism's species, mutant oruncommon allele sequences of an organism's species, sequence insertions,sequence deletions, sequence substitutions, polymorphisms, SNPs, orcombinations thereof. Examples of possible epigenetic elements that canbe assessed using the methods and materials provided herein include,without limitation, methylated DNA. In some cases, an organism can beassessed for one or more of the genetic or epigenetic elements listed inTable 1 using the methods and materials provided herein. When designinga method for detecting a genetic or epigenetic element listed in Table1, a probe nucleic acid can be designed that is complementary to aportion of any of the indicated sequences from Table 1. For example,when designing a method for detecting a single nucleotide polymorphism(SNP) in the human thiopurine S-methyltransferase gene (Ensemble gene IDENSG00000137364; Chromosome 6: 18,128,542-18,155,305 reverse strand), aprobe nucleic acid can be designed that is complementary to a portionthat includes positions 18143896-18143901 of the sequence. In somecases, one or more of the DNA methylation events described elsewhere(e.g., Szyf, Aging Res. Rev., 2(3):299-328 (2003); Di Gioia et al., BMCCancer, 6:89 (2006); Maruya et al., Clin. Cancer Res., 10:3825 (2004);Fischer et al., Lung Cancer, 56:115-123 (2007); Yeo et al., Pathology,37:125-130 (2005); Allen Chan et al., Clin. Chem., 10:1373 (2008); Wanget al., Lung Cancer, 56:289-294 (2007); and Widschwendter et al., PLoSONE, 3(7):e2656 (2008)) can be detected using the methods and materialsprovided herein, for example, to provide information about conditionssuch as carcinogenesis or tumor metastasis.

TABLE 1 Types of genetic or epigenetic elements that can be detected.Possible Associated Organism Genetic or Epigenetic Element ConditionHuman C825T polymorphism of the GNB3 Glioblastoma Human RASSF2A CancersHuman MGMT Cancers Human Cyclins D1 and D2 Cancers (CCDN1 and CCDN2)Human HOXA10 Ovarian cancer Human thiopurine S-methyltransferase Drugtoxicity Human APP, PS1, and PS2 Alzheimer's Disease inheritance of theAPOE4 allele confers increased risk for AD Human MTCYB gene Parkinsondisease Human Congenital heart disease (CHD) Human CHDS1, Chromosome:16; Location: Coronary heart disease 16pter-p13, GeneID: 338334 HumanPARK7, Chromosome: 1; Location: Parkinson disease 1p36.23, GeneID: 11315Human BRCA1, BRCA2 Breast cancer Human BCL2 Cancers Human IL2 CancersHuman CFTR Cystic Fibrosis Human TSC2 Tuberous sclerosis Human APPAlzheimer's Disease Human NPPB Cardiovascular disease Human LEP ObesityHuman OSM Leukemia Human MCM6 lactose intolerance Horse LAMA3 skinblistering disease Cow DGAT1 Milk Production Dog EPM2A Epilepsy

In one embodiment, a method for assessing an organism for a genetic orepigenetic element can include determining whether or not a biologicalsample obtained from the organism contains a target nucleic acid havingthe genetic or epigenetic element of interest. For example, a biologicalsample (e.g., a blood sample to be tested) can be placed in contact withprobe nucleic acid. The probe nucleic acid can be designed to have asingle-stranded portion with a nucleotide sequence that is complementaryto at least a portion of the target nucleic acid to be detected. In thiscase, target nucleic acid present within the sample can hybridize withthe complementary sequence of this single-stranded portion of the probenucleic acid to form a double-stranded section with one strand beingtarget nucleic acid and the other strand being probe nucleic acid. Inaddition, the single-stranded portion of the probe nucleic acid havingthe nucleotide sequence that is complementary to at least a portion ofthe target nucleic acid to be detected can be designed such thathybridization with the target nucleic acid creates a restrictionendonuclease cut site. Thus, target nucleic acid present within thesample can hybridize with the complementary sequence of thesingle-stranded portion of the probe nucleic acid to form adouble-stranded section that creates a cut site for a restrictionendonuclease. This cut site created by the hybridization of targetnucleic acid to probe nucleic acid can be referred to as a recognitionrestriction endonuclease cut site. In addition, a restrictionendonuclease that cleaves nucleic acid at such a recognition restrictionendonuclease cut site can be referred to as a recognition restrictionendonuclease.

The probe nucleic acid also can be designed to contain a restrictionendonuclease. This restriction endonuclease, which can be a component ofthe probe nucleic acid, can be referred to as an amplifying restrictionendonuclease. An amplifying restriction endonuclease is typically adifferent restriction endonuclease than the restriction endonucleasethat is used as a recognition restriction endonuclease. For example,when an EcoRI restriction endonuclease is used as a recognitionrestriction endonuclease, a restriction endonuclease other than an EcoRIrestriction endonuclease (e.g., a Hind III restriction endonuclease) isused as an amplifying restriction endonuclease. Thus, in general, probenucleic acid is designed to contain an amplifying restrictionendonuclease and to have a nucleotide sequence such that the targetnucleic acid can hybridize to the probe nucleic acid and create arecognition restriction endonuclease cut site for a recognitionrestriction endonuclease. In some cases, the probe nucleic acid can beattached to a solid support (e.g., a well of a microtiter plate). Forexample, the probe nucleic acid can be attached to a solid support suchthat cleavage at the recognition restriction endonuclease cut site viathe recognition restriction endonuclease releases a portion of the probenucleic acid that contains the amplifying restriction endonuclease.

After contacting the sample (e.g., a biological sample) that may or maynot contain target nucleic acid with the probe nucleic acid that isattached to a solid support, the target nucleic acid, if present in thesample, can hybridize to the probe nucleic acid and create therecognition restriction endonuclease cut site. At this point, therecognition restriction endonuclease, whether added to the reaction oralready present in the reaction, can cleave the probe nucleic acid atthe recognition restriction endonuclease cut sites that are formed bythe hybridization of target nucleic acid to the probe nucleic acid,thereby releasing the portion of the probe nucleic acid that containsthe amplifying restriction endonuclease from the solid support. Thenumber of amplifying restriction endonuclease-containing portions of theprobe nucleic acid that are released from the solid support can be in anessentially linear relationship (e.g., essentially a one-for-onerelationship) with the number of target nucleic acid molecules thathybridize with the probe nucleic acid to form the recognitionrestriction endonuclease cut site.

The portions of the probe nucleic acid containing the amplifyingrestriction endonuclease that were released from the solid support canbe collected and placed in contact with reporter nucleic acid. Forexample, the released portions of the probe nucleic acid, if present,can be transferred from one well of a microtiter plate (e.g., a 96-wellplate) that contained the probe nucleic acid to another well of amicrotiter plate that contains the reporter nucleic acid. The reporternucleic acid can be designed to have a double-stranded portion with arestriction endonuclease cut site for the amplifying restrictionendonuclease of the probe nucleic acid. This restriction endonucleasecut site for the amplifying restriction endonuclease can be referred toas an amplifying restriction endonuclease cut site. If portions of theprobe nucleic acid containing the amplifying restriction endonucleaseare present and placed in contact with the reporter nucleic acid, thenthe reporter nucleic acid can be cleaved at the amplifying restrictionendonuclease cut site by the amplifying restriction endonuclease. Sincethe amplifying restriction endonucleases of the released portions of theprobe nucleic acid are free to carry out repeated cleavage events, thenumber of reporter nucleic acid molecules that are cleaved can greatlyexceed the number of amplifying restriction endonucleases present in thereaction. For example, the number of cleaved reporter nucleic acidmolecules can greatly exceed (e.g., exponentially exceed) the number ofamplifying restriction endonucleases present in the reaction andtherefore can greatly exceed (e.g., exponentially exceed) the number oftarget nucleic acid molecules that were present in the sample contactedwith the probe nucleic acid. Such a greatly expanded relationship (e.g.,an exponential relationship) can allow very small amounts of targetnucleic acid present in the sample to be readily detected.

After the released portions of the probe nucleic acid, if present, arecontacted with the reporter nucleic acid, the presence or absence ofcleaved reporter nucleic acid can be determined. The presence of cleavedreporter nucleic acid can indicate that the sample contained the targetnucleic acid, thereby indicating that the sample contained the targetgenetic or epigenetic element for which the sample is being tested,while the absence of cleaved reporter nucleic acid can indicate that thesample lacked the target nucleic acid, thereby indicating that thesample lacked the target genetic or epigenetic element for which thesample is being tested. In some cases, the amount of cleaved reporternucleic acid can be determined. In such cases, the amount of cleavedreporter nucleic acid can indicate the amount of target nucleic acidpresent in the sample, which can indicated the relative amount of thegenetic or epigenetic element present in the organism being tested. Astandard curve using known amounts of target nucleic acid can be used toaid in the determination of the amount of target nucleic acid presentwithin a sample. For example, genomic DNA from known heterozygous and/orhomozygous (e.g., homozygous for a particular genetic element beingtested for) organisms can be included in an assay to determine whether agenomic DNA sample from an organism being tested contains zero, one, ortwo copies of a particular target nucleic acid for which the organism isbeing tested based on the amount of cleaved reporter nucleic acid.

In some cases, the reporter nucleic acid can contain a label to aid inthe detection of cleaved reporter nucleic acid. For example, reporternucleic acid can contain a fluorescent label and a quencher such thatcleaved reporter nucleic acid provides a fluorescent signal anduncleaved reporter nucleic acid does not provide a fluorescent signal.In some cases, the reporter nucleic acid can contain a label (e.g., acolorimetric label, a fluorescent label or an enzyme (e.g., a redoxenzyme) such as horse radish peroxidase) and can be attached to a solidsupport (e.g., a well of a microtiter plate). For example, the reporternucleic acid can be attached to a solid support such that cleavage atthe amplifying restriction endonuclease cut site by the amplifyingrestriction endonuclease releases a portion of the reporter nucleic acidthat contains the label. The resulting reaction mixture can be collectedand assessed for the presence, absence, or amount of released portionsof the reporter nucleic acid using the label. For example, the releasedportions of the reporter nucleic acid, if present, can be transferredfrom one well of a microtiter plate (e.g., a 96-well plate) thatcontained the reporter nucleic acid to another well of a microtiterplate, where the transferred material can be assessed for a signal fromthe label.

One example of a method of detecting target nucleic acid that includesusing probe nucleic acid and reporter nucleic acid is set forth inFIG. 1. With reference to FIG. 1, first reaction chamber 100 (e.g., amicrotiter plate well) can contain probe nucleic acid 101. Probe nucleicacid 101 can be attached (e.g., immobilized) to solid support 102 andcan include amplifying restriction endonuclease 103 (Ra). Probe nucleicacid 101 can be attached to solid support 102 such that amplifyingrestriction endonuclease 103 is released from solid support 102 uponcleavage of a nucleic acid component of probe nucleic acid 101. Probenucleic acid 101 can have a single-stranded section having a nucleotidesequence that is complementary to at least a portion of target nucleicacid 104. Probe nucleic acid 101 can be contacted with a sample that mayor may not contain target nucleic acid 104. If target nucleic acid 104is present, at least a portion of target nucleic acid 104 and probenucleic acid 101 can hybridize to form a double-stranded section ofnucleic acid. Such a double-stranded section can contain at least onerecognition restriction endonuclease cut site 105. Addition ofrecognition restriction endonuclease 106 (Rr) to first reaction chamber100 can result in the cleave of probe nucleic acid 101 at recognitionrestriction endonuclease cut site 105 formed by one strand of probenucleic acid and one strand of target nucleic acid, thereby releasingportion 107 of probe nucleic acid 101 from solid support 102. Portion107 can include amplifying restriction endonuclease 103.

The reaction product from first reaction chamber 100 containing releasedportion 107, if target nucleic acid 104 was present, can be transferred(e.g., manually or automatically) to second reaction chamber 120. Secondreaction chamber 120 can contain reporter nucleic acid 121. Reporternucleic acid 121 can be attached (e.g., immobilized) to solid support122 and can include marker (e.g., a label) 123 (M). Reporter nucleicacid 121 can be attached to solid support 122 such that marker 123 isreleased from solid support 122 upon cleavage of a nucleic acidcomponent of reporter nucleic acid 121. Reporter nucleic acid 121 canhave at least one double-stranded portion that contains at least oneamplifying restriction endonuclease cut site 124. Addition of thereaction product from first reaction chamber 100 to second reactionchamber 120 can result in the cleavage of reporter nucleic acid 121 atamplifying restriction endonuclease cut site 124 if the reaction productcontains portion 107. Such cleavage of reporter nucleic acid 121 canresult in the release of portion 127 from solid support 122. Portion 127can include marker 123.

The reaction product from second reaction chamber 120 can be assessed todetermine the presence, absence, or amount of portion 127. The presenceof portion 127 can indicate that the sample contained target nucleicacid 104, while the absence of portion 127 can indicate that the samplelacked target nucleic acid 104. In some cases, the amount of portion 127can be determined. In such cases, the amount of portion 127 can indicatethe amount of target nucleic acid 104 present in the sample. Thepresence, absence, or amount of portion 127 can be determined usingmarker 123, and portion 127 having marker 123 can be distinguished fromuncleaved reporter nucleic acid 121 having marker 123 since, in thisexample, portion 127 is released from solid support 122, while uncleavedreporter nucleic acid 121 remains attached to solid support 122. Forexample, in some cases, the reaction product from second reactionchamber 120 can be transferred to third reaction chamber where thepresence or absence of portion 127 via marker 123 is assessed. Ifportion 127 is present, the amount of portion 127 present can bequantified.

Probe nucleic acid 101 and reporter nucleic acid 121 can have variousconfigurations. For example, with reference to FIG. 1, probe nucleicacid 101 can be designed to have a single nucleic acid strand such thatthe entire nucleic acid component of probe nucleic acid 101 issingle-stranded prior to contact with target nucleic acid 104. Inanother example, with reference to FIG. 2, probe nucleic acid 101 can bedesigned to have first strand 128 and second strand 108. First strand128 can be attached to solid support 102 and can be designed to have asingle-stranded section having a nucleotide sequence that iscomplementary to at least a portion of target nucleic acid 104. Secondstrand 108 can include amplifying restriction endonuclease 103 and canhave a single-stranded section having a nucleotide sequence that canhybridize to first strand 128. In some cases, first strand 128 andsecond strand 108 can be synthesized or obtained separately and thenmixed together to form probe nucleic acid 101. For example, first strand128 can be synthesized, biotinylated, and attached to astreptavidin-coated solid support. After synthesizing the nucleic acidcomponent of second strand 108 and attaching amplifying restrictionendonuclease 103 to the synthesized nucleic acid component, secondstrand 108 can be incubated with first strand 128 to form nucleic acidprobe 101. In some cases, probe nucleic acid 101 can contain more thantwo strands. For example, probe nucleic acid can include first strand150, second strand 152, and third strand 154. In this case, first strand150 can be attached to solid support 102, second strand 152 can behybridized to first strand 150 and can include a single-stranded sectionhaving a nucleotide sequence that is complementary to at least a portionof target nucleic acid 104, and third strand 154 can be hybridized tosecond strand 152 and can be attached to amplifying restrictionendonuclease 103. Similar one, two, three, or more strand configurationscan be used to make reporter nucleic acid.

In another embodiment, a method for detecting target nucleic acid caninclude contacting a sample (e.g., a biological sample to be tested)with probe nucleic acid. The probe nucleic acid can be designed to havea single-stranded portion with a nucleotide sequence that iscomplementary to at least a portion of the target nucleic acid to bedetected. In this case, target nucleic acid present within the samplecan hybridize with the complementary sequence of this single-strandedportion of the probe nucleic acid to form a double-stranded section withone strand being target nucleic acid and the other strand being probenucleic acid. In addition, the single-stranded portion of the probenucleic acid having the nucleotide sequence that is complementary to atleast a portion of the target nucleic acid to be detected can bedesigned such that hybridization with the target nucleic acid creates arecognition restriction endonuclease cut site. Thus, target nucleic acidpresent within the sample can hybridize with the complementary sequenceof the single-stranded portion of the probe nucleic acid to form adouble-stranded section that creates a recognition restrictionendonuclease cut site for a recognition restriction endonuclease. Theprobe nucleic acid also can be designed to contain an amplifyingrestriction endonuclease. Since this method includes the use of two ormore different amplifying restriction endonucleases, the amplifyingrestriction endonuclease that is a component of the probe nucleic acidcan be referred to as a first or an initial amplifying restrictionendonuclease, with additional amplifying restriction endonucleases beingreferred to as second, third, and so on or secondary, tertiary, and soon amplifying restriction endonucleases. This initial amplifyingrestriction endonuclease is typically a different restrictionendonuclease than the restriction endonuclease that is used as arecognition restriction endonuclease. For example, when an EcoRIrestriction endonuclease is used as a recognition restrictionendonuclease, a restriction endonuclease other than an EcoRI restrictionendonuclease (e.g., a Hind III restriction endonuclease) is used as aninitial amplifying restriction endonuclease. Thus, in general, probenucleic acid is designed to contain an initial amplifying restrictionendonuclease and to have a nucleotide sequence such that the targetnucleic acid can hybridize to the probe nucleic acid and create arecognition restriction endonuclease cut site for a recognitionrestriction endonuclease. In some cases, the probe nucleic acid can beattached to a solid support (e.g., a well of a microtiter plate). Forexample, the probe nucleic acid can be attached to a solid support suchthat cleavage at the recognition restriction endonuclease cut site viathe recognition restriction endonuclease releases a portion of the probenucleic acid that contains the initial amplifying restrictionendonuclease.

After contacting the sample that may or may not contain target nucleicacid with the probe nucleic acid that is attached to a solid support,the target nucleic acid, if present in the sample, can hybridize to theprobe nucleic acid and create the recognition restriction endonucleasecut site. At this point, the recognition restriction endonuclease,whether added to the reaction or already present in the reaction, cancleave the probe nucleic acid at the recognition restrictionendonuclease cut sites that are formed by the hybridization of targetnucleic acid to the probe nucleic acid, thereby releasing the portion ofthe probe nucleic acid that contains the initial amplifying restrictionendonuclease from the solid support. The number of initial amplifyingrestriction endonuclease-containing portions of the probe nucleic acidthat are released from the solid support can be in an essentially linearrelationship (e.g., essentially a one-for-one relationship) with thenumber of target nucleic acid molecules that hybridize with the probenucleic acid to form the recognition restriction endonuclease cut site.

The portions of the probe nucleic acid containing the initial amplifyingrestriction endonuclease that were released from the solid support canbe collected and placed in contact with first signal expansion nucleicacid and second signal expansion nucleic acid. The first signalexpansion nucleic acid can be designed to have a double-stranded portionwith a restriction endonuclease cut site for the initial amplifyingrestriction endonuclease of the probe nucleic acid. This restrictionendonuclease cut site for the initial amplifying restrictionendonuclease can be referred to as an initial amplifying restrictionendonuclease cut site. The first signal expansion nucleic acid also canbe designed to contain a secondary amplifying restriction endonuclease.The second signal expansion nucleic acid can be designed to have adouble-stranded portion with a restriction endonuclease cut site for thesecondary amplifying restriction endonuclease of the first signalexpansion nucleic acid. This restriction endonuclease cut site for thesecondary amplifying restriction endonuclease can be referred to as asecondary amplifying restriction endonuclease cut site. The secondsignal expansion nucleic acid also can be designed to contain an initialamplifying restriction endonuclease. For example, when an EcoRIrestriction endonuclease is used as a recognition restrictionendonuclease and a HindIII restriction endonuclease is used as aninitial amplifying restriction endonuclease of the probe nucleic acid, aSmaI restriction endonuclease can be used as a secondary amplifyingrestriction endonuclease of the first signal expansion nucleic acid anda HindIII restriction endonuclease can be used as the initial amplifyingrestriction endonuclease of the second signal expansion nucleic acid.

In some cases, the first signal expansion nucleic acid and second signalexpansion nucleic acid can be attached to a solid support (e.g., a wellof a microtiter plate). For example, the first signal expansion nucleicacid can be attached to a solid support such that cleavage at theinitial amplifying restriction endonuclease cut site via the initialamplifying restriction endonuclease releases a portion of the firstsignal expansion nucleic acid that contains the secondary amplifyingrestriction endonuclease, and the second signal expansion nucleic acidcan be attached to a solid support such that cleavage at the secondaryamplifying restriction endonuclease cut site via the secondaryamplifying restriction endonuclease releases a portion of the secondsignal expansion nucleic acid that contains the initial amplifyingrestriction endonuclease. The first signal expansion nucleic acid can beattached to the same solid support (e.g., two different sub-compartmentsof a larger compartment) that contains the second signal expansionnucleic acid provided that the secondary amplifying restrictionendonuclease of uncleaved first signal expansion nucleic acid is unableto cleave the second signal expansion nucleic acid and provided that theinitial amplifying restriction endonuclease of uncleaved second signalexpansion nucleic acid is unable to cleave the first signal expansionnucleic acid. In some cases, the first signal expansion nucleic acid canbe attached to the same solid support within a joint compartment suchthat the first signal expansion nucleic acid is within a firstcompartment of the joint compartment and the second signal expansionnucleic acid is within a second compartment of the joint compartment. Insuch cases, the secondary amplifying restriction endonuclease ofuncleaved first signal expansion nucleic acid in the first compartmentis unable to cleave the second signal expansion nucleic acid located inthe second compartment, while the secondary amplifying restrictionendonuclease of cleaved first signal expansion nucleic acid is capableof moving (e.g., diffusing) from the first compartment to the secondcompartment to cleave the second signal expansion nucleic acid locatedin the second compartment. In addition, the initial amplifyingrestriction endonuclease of uncleaved second signal expansion nucleicacid in the second compartment is unable to cleave the first signalexpansion nucleic acid located in the first compartment, while theinitial amplifying restriction endonuclease of cleaved second signalexpansion nucleic acid is capable of moving (e.g., diffusing) from thesecond compartment to the first compartment to cleave the first signalexpansion nucleic acid located in the first compartment.

If portions of the probe nucleic acid containing the initial amplifyingrestriction endonuclease are present and placed in contact with thefirst signal expansion nucleic acid, then the first signal expansionnucleic acid can be cleaved at the initial amplifying restrictionendonuclease cut site by the initial amplifying restrictionendonuclease, thereby releasing a portion of the first signal expansionnucleic acid that contains the secondary amplifying restrictionendonuclease from the solid support. The released portions of the firstsignal expansion nucleic acid containing the secondary amplifyingrestriction endonuclease can be free to cleave the second signalexpansion nucleic acid at the secondary amplifying restrictionendonuclease cut site, thereby releasing a portion of the second signalexpansion nucleic acid that contains the initial amplifying restrictionendonuclease from the solid support. Since the initial amplifyingrestriction endonucleases of the released portions of the probe nucleicacid, the initial amplifying restriction endonucleases of the releasedportions of the second signal expansion nucleic acid, and the secondaryamplifying restriction endonucleases of the released portions of thefirst signal expansion nucleic acid are free to carry out repeatedcleavage events, the number of released portions containing the initialamplifying restriction endonucleases is greatly increased from thenumber that were released by the recognition restriction endonuclease.For example, the number of cleaved first signal expansion nucleic acidmolecules can greatly exceed (e.g., exponentially exceed) the number ofreleased portions of the probe nucleic acid, and the number of cleavedsecond signal expansion nucleic acid molecules can greatly exceed (e.g.,exponentially exceed) the number of released portions of the probenucleic acid. Such a greatly expanded relationship (e.g., an exponentialrelationship) can allow very small amounts of target nucleic acidpresent in the sample to be readily detected.

In some cases, this method can be performed with the first signalexpansion nucleic acid being attached to a solid support that isdifferent from the solid support that contains the second signalexpansion nucleic acid. For example, the first signal expansion nucleicacid can be attached to one well of a microtiter plate, while the secondsignal expansion nucleic acid can be attached to a different well of amicrotiter plate. In this case, the resulting reaction material from thewell with the first signal expansion nucleic acid can be collected andtransferred to the well containing the second signal expansion nucleicacid.

The portions of the second signal expansion nucleic acid containing theinitial amplifying restriction endonuclease that were released from thesolid support containing the second signal expansion nucleic acid alongwith any other released portions in this reaction (e.g., the releasedportions of the probe nucleic acid containing the initial amplifyingrestriction endonuclease and the released portions of the first signalexpansion nucleic acid containing the secondary amplifying restrictionendonuclease) can be collected and placed in contact with reporternucleic acid. For example, the released portions, if present, can betransferred from one well of a microtiter plate (e.g., a 96-well plate)that contained the second signal expansion nucleic acid to another wellof a microtiter plate that contains the reporter nucleic acid. Thereporter nucleic acid can be designed to have a double-stranded portionwith a restriction endonuclease cut site for the initial amplifyingrestriction endonuclease. If released portions containing the initialamplifying restriction endonuclease are present and placed in contactwith the reporter nucleic acid, then the reporter nucleic acid can becleaved at the initial amplifying restriction endonuclease cut site bythe initial amplifying restriction endonuclease. Since the initialamplifying restriction endonucleases of the released portions are freeto carry out repeated cleavage events, the number of reporter nucleicacid molecules that are cleaved can greatly exceed the number of initialamplifying restriction endonucleases present in the reaction. Forexample, the number of cleaved reporter nucleic acid molecules cangreatly exceed (e.g., exponentially exceed) the number of initialamplifying restriction endonucleases present in the reaction andtherefore can greatly exceed (e.g., exponentially exceed) the number oftarget nucleic acid molecules that were present in the sample contactedwith the probe nucleic acid. Such a greatly expanded relationship (e.g.,an exponential relationship) can allow very small amounts of targetnucleic acid present in the sample to be readily detected.

After the released portions containing the initial amplifyingrestriction endonuclease, if present, are contacted with the reporternucleic acid, the presence or absence of cleaved reporter nucleic acidcan be determined. The presence of cleaved reporter nucleic acid canindicate that the sample contained the target nucleic acid, therebyindicating that the sample contained the target genetic or epigeneticelement for which the sample is being tested, while the absence ofcleaved reporter nucleic acid can indicate that the sample lacked thetarget nucleic acid, thereby indicating that the sample lacked thetarget genetic or epigenetic element for which the sample is beingtested.

In some cases, the amount of cleaved reporter nucleic acid can bedetermined. In such cases, the amount of cleaved reporter nucleic acidcan indicate the amount of target nucleic acid present in the sample,which can indicated the relative amount of the genetic or epigeneticelement present in the organism being tested. A standard curve usingknown amounts of target nucleic acid can be used to aid in thedetermination of the amount of target nucleic acid present within asample. For example, genomic DNA from known heterozygous and/orhomozygous (e.g., homozygous for a particular genetic element beingtested for) organisms can be included in an assay to determine whether agenomic DNA sample from an organism being tested contains zero, one, ortwo copies of a particular target nucleic acid for which the organism isbeing tested based on the amount of cleaved reporter nucleic acid.

In some cases, the reporter nucleic acid can contain a label to aid inthe detection of cleaved reporter nucleic acid. For example, reporternucleic acid can contain a fluorescent label and a quencher such thatcleaved reporter nucleic acid provides a fluorescent signal anduncleaved reporter nucleic acid does not provide a fluorescent signal.In some cases, the reporter nucleic acid can contain a label (e.g., acolorimetric label, fluorescent label or an enzyme such as horse radishperoxidase) and can be attached to a solid support (e.g., a well of amicrotiter plate). For example, the reporter nucleic acid can beattached to a solid support such that cleavage at the initial amplifyingrestriction endonuclease cut site by the initial amplifying restrictionendonuclease releases a portion of the reporter nucleic acid thatcontains the label. The resulting reaction mixture can be collected andassessed for the presence, absence, or amount of released portions ofthe reporter nucleic acid using the label. For example, the releasedportions of the reporter nucleic acid, if present, can be transferredfrom one well of a microtiter plate (e.g., a 96-well plate) thatcontained the reporter nucleic acid to another well of a microtiterplate, where the transferred material can be assessed for a signal fromthe label.

In some cases, the presence or absence of cleaved first signal expansionnucleic acid, cleaved second signal expansion nucleic acid, or both canbe determined. The presence of such cleaved nucleic acid can indicatethat the sample contained the target nucleic acid, thereby indicatingthat the sample contained the target genetic or epigenetic element forwhich the sample is being tested, while the absence of such cleavednucleic acid can indicate that the sample lacked the target nucleicacid, thereby indicating that the sample lacked the target genetic orepigenetic element for which the sample is being tested. In some cases,the amount of cleaved first signal expansion nucleic acid, cleavedsecond signal expansion nucleic acid, or both can be determined. In suchcases, the amount of cleaved nucleic acid can indicate the amount oftarget nucleic acid present in the sample, which can indicated therelative amount of the genetic or epigenetic element present in theorganism being tested. In these cases, the use of cleaved first signalexpansion nucleic acid, cleaved second signal expansion nucleic acid, orboth to assess the sample for target nucleic acid can be in addition tothe use of a separate reporter nucleic acid step or can replace the useof a separate reporter nucleic acid step. In some cases, the firstsignal expansion nucleic acid, the second signal expansion nucleic acid,or both can be labeled in a manner similar to that described herein forthe reporter nucleic acid to aid in detection. When the presence,absence, or amount of cleaved first signal expansion nucleic acid,cleaved second signal expansion nucleic acid, or both are determined toassess the sample for target nucleic acid, the first signal expansionnucleic acid can be referred to as a first reporter nucleic acid and thesecond signal expansion nucleic acid can be referred to as a secondreporter nucleic acid even though they include amplifying restrictionendonucleases. A standard curve using known amounts of target nucleicacid can be used to aid in the determination of the amount of targetnucleic acid present within a sample. For example, genomic DNA fromknown heterozygous and/or homozygous (e.g., homozygous for a particulargenetic element being tested for) organisms can be included in an assayto determine whether a genomic DNA sample from an organism being testedcontains zero, one, or two copies of a particular target nucleic acidfor which the organism is being tested based on the amount of cleavedfirst signal expansion nucleic acid, cleaved second signal expansionnucleic acid, or both.

Examples of a method of detecting target nucleic acid that includesusing probe nucleic acid, first signal expansion nucleic acid, secondsignal expansion nucleic acid, and reporter nucleic acid are set forthin FIGS. 3-5. With reference to FIG. 3, first reaction chamber 200(e.g., a microtiter plate well) can contain probe nucleic acid 201.Probe nucleic acid 201 can be attached (e.g., immobilized) to solidsupport 202 and can include initial amplifying restriction endonuclease203 (Ra). Probe nucleic acid 201 can be attached to solid support 202such that initial amplifying restriction endonuclease 203 is releasedfrom solid support 202 upon cleavage of a nucleic acid component ofprobe nucleic acid 201. Probe nucleic acid 201 can have asingle-stranded section having a nucleotide sequence that iscomplementary to at least a portion of target nucleic acid 204. Probenucleic acid 201 can be contacted with a sample that may or may notcontain target nucleic acid 204. If target nucleic acid 204 is present,at least a portion of target nucleic acid 204 and probe nucleic acid 201can hybridize to form a double-stranded section of nucleic acid. Such adouble-stranded section can contain at least one recognition restrictionendonuclease cut site 205. Addition of recognition restrictionendonuclease 206 (Rr) to first reaction chamber 200 can result in thecleavage of probe nucleic acid 201 at recognition restrictionendonuclease cut site 205 formed by one strand of probe nucleic acid andone strand of target nucleic acid, thereby releasing portion 207 ofprobe nucleic acid 201 from solid support 202. Portion 207 can includeinitial amplifying restriction endonuclease 203.

The reaction product from first reaction chamber 200 containing releasedportion 207, if target nucleic acid 204 was present, can be transferred(e.g., manually or automatically) to second reaction chamber 220. Secondreaction chamber 220 can contain first signal expansion nucleic acid 226and second signal expansion nucleic acid 225. First signal expansionnucleic acid 226 can have at least one double-stranded portion thatcontains at least one initial amplifying restriction endonuclease cutsite 230. First signal expansion nucleic acid 226 can be attached (e.g.,immobilized) to solid support 222 and can include secondary amplifyingrestriction endonuclease 223 (Rb). First signal expansion nucleic acid226 can be attached to solid support 222 such that portion 234containing secondary amplifying restriction endonuclease 223 is releasedfrom solid support 222 upon cleavage of first signal expansion nucleicacid 226 at initial amplifying restriction endonuclease cut site 230.For clarity, frame E of FIG. 3 omits depicting one strand from thecleaved versions of first signal expansion nucleic acid 226 and secondsignal expansion nucleic acid 225.

Second signal expansion nucleic acid 225 can have at least onedouble-stranded portion that contains at least one secondary amplifyingrestriction endonuclease cut site 232. Second signal expansion nucleicacid 225 can be attached (e.g., immobilized) to solid support 222 andcan include initial amplifying restriction endonuclease 224. Secondsignal expansion nucleic acid 225 can be attached to solid support 222such that portion 236 containing initial amplifying restrictionendonuclease 224 is released from solid support 222 upon cleavage ofsecond signal expansion nucleic acid 225 at secondary amplifyingrestriction endonuclease cut site 232. Initial amplifying restrictionendonuclease 203 of probe nucleic acid 201 and initial amplifyingrestriction endonuclease 224 of second signal expansion nucleic acid 225can be the same restriction endonuclease. For example, both can be anEcoRI restriction endonuclease.

Addition of the reaction product from first reaction chamber 200 tosecond reaction chamber 220 can result in the cleavage of first signalexpansion nucleic acid 226 at initial amplifying restrictionendonuclease cut site 230 if the reaction product contains portion 207.Such cleavage of first signal expansion nucleic acid 226 can result inthe release of portion 234 from solid support 222. Portion 234, whichcan include secondary amplifying restriction endonuclease 223, canresult in the cleavage of second signal expansion nucleic acid 225 atsecondary amplifying restriction endonuclease cut site 232. Suchcleavage of second signal expansion nucleic acid 225 can result in therelease of portion 236 from solid support 222. Thus, this reaction canresult in the accumulation of released portions 234 and 236.

The reaction product from second reaction chamber 220 containingreleased portion 207, released portion 234, and released portion 236, iftarget nucleic acid 204 was present, can be transferred (e.g., manuallyor automatically) to third reaction chamber 240. Third reaction chamber240 can contain reporter nucleic acid 241. Reporter nucleic acid 241 canbe attached (e.g., immobilized) to solid support 242 and can includemarker (e.g., a label) 243 (M). Reporter nucleic acid 241 can beattached to solid support 242 such that marker 243 is released fromsolid support 242 upon cleavage of a nucleic acid component of reporternucleic acid 241. Reporter nucleic acid 241 can have at least onedouble-stranded portion that contains at least one initial amplifyingrestriction endonuclease cut site 246. Addition of the reaction productfrom second reaction chamber 220 to third reaction chamber 240 canresult in the cleavage of reporter nucleic acid 241 at initialamplifying restriction endonuclease cut site 246 if the reaction productcontains portion 207 and portion 236. In some cases, reporter nucleicacid 241 can include at least one double-stranded portion that containsat least one cut site for secondary amplifying restriction endonuclease223. In such cases, addition of the reaction product from secondreaction chamber 220 to third reaction chamber 240 can result in thecleavage of reporter nucleic acid 241 at the cut site for secondaryamplifying restriction endonuclease 223 if the reaction product containsportion 234. Cleavage of reporter nucleic acid 241 can result in therelease of portion 247 from solid support 242. Portion 247 can includemarker 243.

The reaction product from third reaction chamber 240 can be assessed todetermine the presence, absence, or amount of portion 247. The presenceof portion 247 can indicate that the sample contained target nucleicacid 204, while the absence of portion 247 can indicate that the samplelacked target nucleic acid 204. In some cases, the amount of portion 247can be determined. In such cases, the amount of portion 247 can indicatethe amount of target nucleic acid 204 present in the sample. Thepresence, absence, or amount of portion 247 can be determined usingmarker 243, and portion 247 having marker 243 can be distinguished fromuncleaved reporter nucleic acid 241 having marker 243 since, in thisexample, portion 247 is released from solid support 242, while uncleavedreporter nucleic acid 241 remains attached to solid support 242. Forexample, in some cases, the reaction product from third reaction chamber24 can be transferred to fourth reaction chamber where the presence orabsence of portion 247 via marker 243 is assessed. If portion 347 ispresent, the amount of portion 247 present can be quantified.

In some cases and with reference to FIGS. 4 and 5, first signalexpansion nucleic acid 226 can include marker (e.g., a label) 243 (M)and second signal expansion nucleic acid 225 can include marker (e.g., alabel) 243 (M). In such cases, cleavage of first signal expansionnucleic acid 226 and cleavage of second signal expansion nucleic acid225 can be assessed using marker 243 to determine the presence, absence,or amount of target nucleic acid within a sample. For example, detector250 can be used to detect marker 243 released from solid support 222.

Probe nucleic acid 201, first signal expansion nucleic acid 226, secondsignal expansion nucleic acid 225, and reporter nucleic acid 241 canhave various configurations. For example, with reference to FIG. 3,probe nucleic acid 201 can be designed to have a single nucleic acidstrand such that the entire nucleic acid component of probe nucleic acid201 is single-stranded prior to contact with target nucleic acid 204. Inanother example, probe nucleic acid 201 can be designed in a manner likeprobe nucleic acid 101 to have two or more strands. See, e.g., FIG. 2.For example, probe nucleic acid 201 can have a first strand and a secondstrand. The first strand can be attached to a solid support and can bedesigned to have a single-stranded section having a nucleotide sequencethat is complementary to at least a portion of target nucleic acid. Thesecond strand can include an initial amplifying restriction endonucleaseand can have a single-stranded section having a nucleotide sequence thatcan hybridize to the first strand. In some cases, the first strand andsecond strand can be synthesized or obtained separately and then mixedtogether to form probe nucleic acid 201. For example, the first strandcan be synthesized, biotinylated, and attached to a streptavidin-coatedsolid support. After synthesizing the nucleic acid component of thesecond strand and attaching an initial amplifying restrictionendonuclease to the synthesized nucleic acid component, the secondstrand can be incubated with the first strand to form nucleic acid probe201. In some cases, probe nucleic acid 201 can contain more than twostrands. For example, probe nucleic acid can include a first strand, asecond strand, and a third strand. In this case, the first strand can beattached to a solid support, the second strand can be hybridized to thefirst strand and can include a single-stranded section having anucleotide sequence that is complementary to at least a portion oftarget nucleic acid, and the third strand can be hybridized to thesecond strand and can be attached to an initial amplifying restrictionendonuclease. Similar one, two, three, or more strand configurations canbe used to make first signal expansion nucleic acid, second signalexpansion nucleic acid, or reporter nucleic acid. For example, firstsignal expansion nucleic acid and second signal expansion nucleic acidcan be designed to have a configuration as shown in FIG. 4 or 5.

Probe nucleic acid described herein typically includes at least onesingle-stranded DNA section that is designed to hybridize with a desiredtarget nucleic acid and thereby create a recognition restrictionendonuclease cut site. The other portions of the probe nucleic acid caninclude DNA, RNA, or other molecules. For example, probe nucleic acidcan include biotin such that the probe nucleic acid can be attached to astreptavidin-coated solid support. In some cases, the single-strandedsection of the probe nucleic acid that is designed to hybridize with adesired target nucleic acid and create a recognition restrictionendonuclease cut site can be RNA or a nucleic acid analog (e.g., apeptide nucleic acid (PNA)) provided that such a single-stranded sectioncan (i) hybridize with the desired target nucleic acid and (ii) create arecognition restriction endonuclease cut site with the complementarytarget nucleic acid sequence that is capable of being cleaved by therecognition restriction endonuclease. Examples of restrictionendonucleases that can be used as recognition restriction endonucleasesto cleave a recognition restriction endonuclease cut site that iscreated between an RNA section of the probe nucleic acid and a DNAsection of the target nucleic acid include, without limitation, HhaI,AluI, TaqI, HaeIII, EcoRI, HindII, SalI, and MspI restrictionendonucleases.

Probe nucleic acid described herein can be any length provided that thesingle-stranded section of the probe nucleic acid that is designed tohybridize with a desired target nucleic acid is capable of hybridizingto the target nucleic acid and provided that the amplifying restrictionendonuclease of the probe nucleic acid is capable of cleaving itsamplifying restriction endonuclease cut site after the probe nucleicacid is cleaved by a recognition restriction endonuclease. In general,the single-stranded section of the probe nucleic acid that is designedto hybridize with a desired target nucleic acid can be between about 10and about 500 or more nucleotides (e.g., between about 10 and about 400nucleotides, between about 10 and about 300 nucleotides, between about10 and about 200 nucleotides, between about 10 and about 100nucleotides, between about 10 and about 50 nucleotides, between about 10and about 25 nucleotides, between about 20 and about 500 nucleotides,between about 30 and about 500 nucleotides, between about 40 and about500 nucleotides, between about 50 and about 500 nucleotides, betweenabout 15 and about 50 nucleotides, between about 15 and about 25nucleotides, between about 20 and about 50 nucleotides, between about 18and about 25 nucleotides, between about 20 and about 60 nucleotides,between about 25 and about 55 nucleotides, between about 30 and about 50nucleotides, between about 35 and about 45 nucleotides, or between about38 and about 42 nucleotides) in length. The recognition restrictionendonuclease cut site that will be created by the hybridization oftarget nucleic acid to this single-stranded section of the probe nucleicacid can be located at any position alone the single-stranded section.For example, the recognition restriction endonuclease cut site to becreated can be towards the 5′ end, towards the '3 end, or near thecenter of the single-stranded section of the probe nucleic acid. Ingeneral, the overall length of the probe nucleic acid described hereincan be between about 10 and about 2500 or more nucleotides (e.g.,between about 10 and about 2000 nucleotides, between about 10 and about1000 nucleotides, between about 10 and about 500 nucleotides, betweenabout 10 and about 400 nucleotides, between about 10 and about 300nucleotides, between about 10 and about 200 nucleotides, between about10 and about 100 nucleotides, between about 10 and about 50 nucleotides,between about 10 and about 25 nucleotides, between about 20 and about500 nucleotides, between about 30 and about 500 nucleotides, betweenabout 40 and about 500 nucleotides, between about 50 and about 500nucleotides, between about 75 and about 500 nucleotides, between about100 and about 500 nucleotides, between about 150 and about 500nucleotides, between about 15 and about 50 nucleotides, between about 15and about 25 nucleotides, between about 20 and about 50 nucleotides,between about 18 and about 25 nucleotides, between about 20 and about 60nucleotides, between about 25 and about 55 nucleotides, between about 30and about 50 nucleotides, between about 35 and about 45 nucleotides, orbetween about 38 and about 42 nucleotides) in length.

The recognition restriction endonuclease cut site to be created byhybridization of target nucleic acid to the probe nucleic acid can be acut site of any type of restriction endonuclease. In addition, any typeof restriction endonuclease can be used as a recognition restrictionendonuclease to cleave probe nucleic acid upon target nucleic acidhybridization. Examples of restriction endonucleases that can be used asrecognition restriction endonucleases include, without limitation,EcoRI, EcoRII, BamHI, HindIII, TaqI, NotI, HinfI, Sau3A, PovII, SmaI,HaeIII, HgaI, AluI, EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI, SphI,StuI, XbaI, AarI, BanII, BseGI, BspPI, CfrI, EcoNI, Hsp92II, NlaIV,RsaI, TaiI, AasI, BbsI, BseJI, BspTI, ClaI, EcoO109I, I-PpoI, NmuCI,RsrII, TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI, KasI, Acc65I, BbvCI,BseMI, BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI, AccB7I, BbvI, BseMII,BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI, BceAI, BseNI, BsrGI,CspI, Esp3I, KspAI, NsiI, SapI, and TauI restriction endonucleases. Insome cases, nucleic acid encoding a naturally-occurring restrictionendonuclease can be genetically engineered to create a modifiedrestriction endonuclease that has the ability to recognize a particularcut site. Common computer algorithms can be used to locate restrictionendonuclease cut sites along the nucleotide sequence of any desiredtarget nucleic acid. Once located, the sequence of the restrictionendonuclease cut site along with additional flanking sequence (e.g., 5′flanking sequence, 3′ flanking sequence, or both 5′ and 3′ flankingsequence) can be used to design the complementary sequence of the probenucleic acid that is used to hybridize to the target nucleic acid andcreate the recognition restriction endonuclease cut site upon targetnucleic acid hybridization. In some cases, a probe nucleic acid can bedesigned to have the restriction endonuclease cut site located in themiddle or near the middle such that the restriction endonuclease cutsite has both 5′ and 3′ flanking sequences that are complementary to thetarget nucleic acid.

In some cases, the probe nucleic acid is designed to have asingle-stranded section that is designed to hybridize with desiredtarget nucleic acid and to form a recognition restriction endonucleasecut site upon target nucleic acid hybridization such that target nucleicacid containing the particular genetic or epigenetic element beingtested for hybridizes to the probe nucleic acid and, together with theprobe nucleic acid, is cleaved by the recognition restrictionendonuclease, thereby releasing a portion of the probe nucleic acid,while nucleic acid lacking the particular genetic or epigenetic elementbeing tested for does not result in the formation of cleaved probenucleic acid even though such nucleic acid lacking the genetic orepigenetic element may hybridize with the probe nucleic acid. Forexample, a probe nucleic acid can be designed to contain asingle-stranded portion that is designed to hybridize with a targetnucleic acid containing a particular SNP sequence and to form arecognition restriction endonuclease cut site at the location of theparticular SNP sequence upon target nucleic acid hybridization. In suchcases, the target nucleic acid containing the particular SNP sequencebeing tested for can hybridize to the probe nucleic acid and, togetherwith the probe nucleic acid, can be cleaved by the recognitionrestriction endonuclease, thereby releasing a portion of the probenucleic acid, while nucleic acid lacking the particular SNP sequencebeing tested for can fail to result in the formation of cleaved probenucleic acid even though such nucleic acid lacking the particular SNPsequence may hybridize with the probe nucleic acid (see, e.g., FIG. 10).In these cases, the recognition restriction endonuclease can be used todifferentiate between target nucleic acid containing the particulargenetic or epigenetic element being tested for and other nucleic acidsthat lack the particular genetic or epigenetic element being tested foreven though such other nucleic acids may hybridize with the probenucleic acid. In some cases, the difference between these other nucleicacids and the target nucleic acid being tested for can be a singlenucleotide. For example, ApoI can be used as a recognition restrictionendonuclease to differentiate between a target nucleic acid containing a5′-AAATTC-3′ sequence and other nucleic acids that simply have a5′-AAATTA-3′ sequence in place of the 5′-AAATTC-3′ sequence.

In some cases, probe nucleic acid can be designed for use with arecognition restriction endonuclease that has separate recognition andcleavage sites such as an FokI restriction endonuclease (FIG. 11). FokIrecognizes a specific 5-base site (5′-GGATG-3′), but it cleaves thedouble stranded nucleic acid at a position nine bases downstream of therecognition site provided that these nine bases form perfectly matcheddouble-stranded sequence (FIG. 11). Other examples of such restrictionendonucleases include, without limitation, AlwI, MnlI CspCI, AjuI, AloI,PpiI, PsrI, and AarI. Probe nucleic acid designed for use with arecognition restriction endonuclease that has separate recognition andcleavage sites can be used to detect any SNP of interest, includingthose that do not change a known restriction site with respect to, forexample, corresponding wild-type sequences. In such cases, probe nucleicacid can be designed to contain a double stranded portion between 10 and100 bp in length (e.g., 10 and 75, 10 and 50, 10 and 40, 10 and 30, 20and 100, 30 and 100, 15 and 75, 15 and 50, 15 and 40, 20 and 50, or 20and 40 bp in length) that has the recognition site of a recognitionrestriction endonuclease that has separate recognition and cleavagesites (e.g., FokI) adjacent to a single stranded portion 10 and 100nucleotides in length (e.g., 10 and 75, 10 and 50, 10 and 40, 10 and 30,20 and 100, 30 and 100, 15 and 75, 15 and 50, 15 and 40, 20 and 50, or20 and 40 nucleotides in length) that is designed to have a sequencecomplementary to a desired target nucleic acid (e.g., a wild-type targetnucleic acid or a target nucleic acid containing a SNP) such thathybridization of the desired target nucleic acid creates the cleavagesite of the recognition restriction endonuclease. Probe nucleic acidcontaining any hybridized nucleic acid can be subjected to blunting andligation reactions. For example, T4 DNA polymerase (or a blunting kitcontaining this enzyme) can be used to remove free single-stranded endsof nucleic acid hybridized to probe nucleic acid. T4 DNA polymerase canconvert DNA with single-stranded 5′or 3′ overhangs to 5′ phosphorylated,blunt-ended DNA for efficient blunt-end ligation. A DNA ligase (e.g., E.coli DNA ligase) can be used subsequently (or simultaneously with T4 DNApolymerase) to ligate the blunted hybridized nucleic acid to theadjacent strand of probe nucleic acid (see, e.g., FIG. 12).

If the desired target nucleic acid is present in a sample being tested,hybridizes to the single stranded portion of probe nucleic acid to formthe cleavage site of the recognition restriction endonuclease that hasseparate recognition and cleavage sites, and is ligated to the adjacentstrand of probe nucleic acid, then the recognition restrictionendonuclease can cleave the probe nucleic acid:target nucleic acidhybrid (see, e.g., FIG. 12). Such cleavage can be detected using themethods and materials provided herein. For example, the portion ofcleaved probe nucleic acid containing the amplifying restrictionendonuclease can be allowed to cleave reporter nucleic acid as describedherein.

With reference to FIG. 12, probe nucleic acid can be designed to have adouble-stranded DNA section between 10 and 100 bp in length (e.g., 10and 75, 10 and 50, 10 and 40, 10 and 30, 20 and 100, 30 and 100, 15 and75, 15 and 50, 15 and 40, 20 and 50, or 20 and 40 bp in length) carryingthe FokI recognition site (GGATG) at the free end and a single-strandedDNA section 10 and 100 nucleotides in length (e.g., 10 and 75, 10 and50, 10 and 40, 10 and 30, 20 and 100, 30 and 100, 15 and 75, 15 and 50,15 and 40, 20 and 50, or 20 and 40 nucleotides in length) that iscomplementary to a target nucleic acid DNA fragment (in this example, awild-type nucleic acid sequence of interest). The single-strandedsequence of the probe nucleic acid is designed such that a potential SNPlocation is positioned at the potential FokI cleavage site, which isexactly nine nucleotides away from the FokI recognition site. The probenucleic acid is allowed to hybridize to nucleic acid present in thesample being tested, and any free single-stranded ends of hybridizednucleic acid from the sample being tested are removed using T4 DNApolymerase (or a blunting kit containing this enzyme). A DNA ligase(e.g., E. coli DNA ligase) is used to ligate any blunted hybridizednucleic acid from the sample being tested to the adjacent available endof a strand of the probe nucleic acid. If the desired target nucleicacid is present in the sample, a target nucleic acid:probe nucleic acidhybrid is formed such that the hybrid contains both double-stranded FokIrecognition and cleavage sites. At this point, FokI can cleave thetarget nucleic acid:probe nucleic acid hybrid, which can be detected asdescribed herein. If any blunted hybridized nucleic acid from the samplebeing tested contains a SNP such that a mismatch exists with the probenucleic acid at the cleavage site, then such probe nucleic acid are notcleaved.

In some cases, an assay or kit provided herein can have one probenucleic acid designed to detect target nucleic acid having an un-mutatedsequence (e.g., a wild-type sequence) and another probe nucleic aciddesigned to detect target nucleic acid having a mutated version of thesequence (e.g., a sequence containing a SNP). Comparison of signals formutated versus un-mutated target nucleic acids can provide informationabout the homozygosity and heterozygosity of the corresponding genotypein terms of the allele of interest.

In general, probe nucleic acid can be designed to have a single-strandedsection that is designed to hybridize with desired target nucleic acidand to form a single recognition restriction endonuclease cut site upontarget nucleic acid hybridization. In some cases, probe nucleic acid canbe designed to have a single-stranded section that is designed tohybridize with desired target nucleic acid and to form more than one(e.g., two, three, four, five, six, seven, eight, nine, ten, or more)recognition restriction endonuclease cut site upon target nucleic acidhybridization. When more than one recognition restriction endonucleasecut site is used, the multiple recognition restriction endonuclease cutsites can be cut sites for the same restriction endonuclease or cutsites for different restriction endonucleases. For example, probenucleic acid can be designed to have a single-stranded section that isdesigned to hybridize with desired target nucleic acid and to form onerecognition restriction endonuclease cut site for an EcoRI recognitionrestriction endonuclease and one recognition restriction endonucleasecut site for an XbaI recognition restriction endonuclease upon targetnucleic acid hybridization. In such cases, each recognition restrictionendonuclease can be used individually or in combination (e.g., as amixture) to cleave probe nucleic acid that hybridized to target nucleicacid and formed the corresponding recognition restriction endonucleasecut site via such hybridization.

Probe nucleic acid can be designed such that any target nucleic acidcontaining a genetic or epigenetic element can be detected. Examples oftarget nucleic acid that can be detected using the methods and materialsprovided herein include, without limitation, genomic DNA, RNA, cDNA,methylated DNA, and combinations thereof. In some cases such as thoseinvolving assessing a biological sample for a genetic or epigeneticelement in RNA, the target nucleic acid can be an RNA or a cDNAgenerated from an RNA. When detecting an RNA target nucleic acid,restriction endonucleases having the ability to cleave a recognitionrestriction endonuclease cut site that is created between a DNA sectionof the probe nucleic acid and the RNA target nucleic acid can be used asrecognition restriction endonucleases. Examples of such restrictionendonucleases include, without limitation, HhaI, AluI, TaqI, HaeIII,EcoRI, HindII, SalI, and MspI restriction endonucleases. When detectingmethylated target nucleic acid (e.g., a methylated DNA), restrictionendonucleases having the ability to cleave a recognition restrictionendonuclease cut site that includes a methylated nucleotide to beassessed can be used as recognition restriction endonucleases. Examplesof restriction endonucleases having the ability to recognize methylatednucleotides include, without limitation, DpnI, GlaI, HpaII, MspI, AciI,HhaI, and SssI restriction endonucleases. In such cases, a control caninclude detecting the same target nucleic acid without the methylatednucleotide. In some cases, a combination of methylation insensitive andmethylation sensitive restriction endonucleases can be used to assess asample for methylated target nucleic acid. For example, similargeneration of cleavage products using both methylation insensitive andmethylation sensitive restriction endonucleases designed for the samesite can indicate that the target nucleic acid lacks methylation at thatsite, while an increased level of cleavage products using a methylationinsensitive restriction endonuclease as compared to the level generatedusing a methylation sensitive restriction endonuclease designed for thesame site can indicate that the target nucleic acid is methylated atthat site (see, e.g., FIG. 8).

Any appropriate pair of methylation sensitive and methylationinsensitive isoschizomers can be used as described herein. Examples ofsuch recognition restriction endonuclease pairs include, withoutlimitation, the MspI/HpaII pair that cut CCGG sites, with HpaII beingsensitive to methylation of the second C (blocked by CCmGG) and theEcoRII/BstN1 pair that cut CCNGG sites, with BstN1 beingmethylation-sensitive. There are more than 300 methylation sensitiverestriction endonucleases known that can be used as described herein,and about 30 of them have methylation insensitive isoschizomers(McClelland et al., Nucleic Acids Res., 22:3640-3659 (1994)).

In some cases, the presence, absence, or amount of target methylated DNAcan be determined using methylation sensitive recognition restrictionendonucleases as opposed to a pair of methylation sensitive andmethylation insensitive isoschizomers (see, e.g., FIG. 9). For example,target DNA can be contacted with the same probe nucleic acid in twodifferent compartments or wells (e.g., compartment 1 and compartment 2).A first recognition restriction endonuclease (e.g., Rr1), which cleavesa cut site (e.g., cut site A) that does not contain any C nucleotidesand thus can not be methylated, can be added to compartment 1. Uponhybridization with the target DNA, all probe-target hybrids are cleavedby Rr1, and the signal from compartment 1 corresponds to the totalamount of the target DNA in the sample. A second recognition restrictionendonuclease (e.g., Rr2), which is methylation-sensitive and can onlycleave a different site (e.g., site B) if it is unmethylated, can beadded to compartment 2. Upon hybridization with the target DNA,probe-target hybrids are cleaved by Rr2 only if site B is unmethylated,and the signal from compartment 2 corresponds only to the unmethylatedtarget DNA within the sample. Signal detection for both compartments canbe carried out as described herein. The resultant signal fromcompartment 1 can correspond to the total amount of target DNA, whilethe compartment 2 signal can correspond only to the amount ofunmethylated target DNA. Thus, the amount of methylated target can becalculated by subtracting the compartment 2 signal from the compartment1 signal.

The nucleotide sequence of target nucleic acid to be detected can beobtained from, for example, common nucleic acid sequence databases suchas GenBank® (e.g., the SNP database of GenBank®). A portion of targetnucleic acid sequence can be selected using a computer-based program.For example, a computer-based program can be used to detect restrictionendonuclease cut sites within a portion of target nucleic acid (e.g., atthe location of a genetic or epigenetic element whether the genetic orepigenetic element is a single nucleotide element or a larger nucleotidesequence element such as a 5, 10, 15, 20, 50, 100, or more nucleotideinsertion). Such information can be used to design probe nucleic acidsuch that the single-stranded section creates at least one recognitionrestriction endonuclease cut site upon hybridization of the targetnucleic acid. In some cases, bioinformatics computer-based programs andtools can be used to assist in the design of probe nucleic acid. Forexample, computer programs (e.g., BLAST® and alignment programs) andcomputer databases (e.g., GenBank®) can be used to indentify nucleicacid sequences of a particular organism's genome (e.g., sequences fromparticular genes, coding sequences, promotors, enhancers, oruntranslated regions). In addition, computer programs such as CLCWorkbench or Vector NTI (Invitrogen) can be used to identify thelocation of restriction endonuclease cut sites within a particularnucleic acid sequence. In some cases, sequence analysis computerprograms can be used to identify sequences with limited or an absence ofrepeats, a presence of high sequence complexity of a potentialrecognition restriction endonuclease cut site, and/or limited or anabsence of hairpin structures. Identification of such sequences can helpreduce the risk of probe self-hybridization and potentially unintendedcutting by a recognition endonuclease.

Any appropriate method can be used to obtain the nucleic acid componentof the probe nucleic acid. For example, common molecular cloning andchemical nucleic acid synthesis techniques can be used to obtain thenucleic acid component of the probe nucleic acid. In some cases, thenucleic acid component of the probe nucleic acid can be synthesizedusing commercially available automated oligonucleotide synthesizers suchas those available from Applied Biosystems (Foster City, Calif.). Insome cases, probe nucleic acids can be synthesized de novo using any ofa number of procedures widely available in the art. Examples of suchmethods of synthesis include, without limitation, the β-cyanoethylphosphoramidite method (Beaucage et al., Tet. Let., 22:1859-1862 (1981))and the nucleoside H-phosphonate method (Garegg et al., Tet. Let.,27:4051-4054 (1986); Froehler et al., Nucl. Acid Res., 14:5399-5407(1986); Garegg et al., Tet. Let., 27:4055-4058 (1986); and Gaffney etal., Tet. Let., 29:2619-2622 (1988)). These methods can be performed bya variety of commercially-available automated oligonucleotidesynthesizers. In some cases, recombinant nucleic acid techniques such asPCR and those that include using restriction enzyme digestion andligation of existing nucleic acid sequences (e.g., genomic DNA or cDNA)can be used to obtain the nucleic acid component of the probe nucleicacid.

Probe nucleic acid described herein can be attached to a solid support.Examples of solid supports include, without limitation, a well of amicrotiter plate (e.g., a 96-well microtiter plate or ELISA plate),beads (e.g., magnetic, glass, plastic, or gold-coated beads), slides(e.g., glass or gold-coated slides), micro- or nano-particles (e.g.,carbon nanotubes), platinum solid supports, palladium solid supports,and a surface of a chamber or channel within a microfluidic device. Insome cases, a solid support can be a silicon oxide-based solid support,a plastic polymer-based solid support (e.g., a nylon, nitrocellulose, orpolyvinylidene fluoride-based solid support), or a biopolymer-based(e.g., a cross-linked dextran or cellulose-based solid support) solidsupport. Probe nucleic acid can be directly or indirectly attached to asolid support. For example, biotin can be a component of the probenucleic acid, and the probe nucleic acid containing biotin can beindirectly attached to a solid support that is coated with streptavidinvia a biotin-streptavidin interaction. In some cases, probe nucleic acidcan be attached to a solid support via a covalent or non-covalentinteraction. For example, probe nucleic acid can be covalently attachedto magnetic beads as described elsewhere (Albretsen et al., Anal.Biochem., 189(1):40-50 (1990)).

Probe nucleic acid can be designed to contain any type of restrictionendonuclease as an amplifying restriction endonuclease. In general, anamplifying restriction endonuclease of the probe nucleic acid istypically a different restriction endonuclease than the restrictionendonuclease that is used as a recognition restriction endonuclease. Forexample, when an EcoRI restriction endonuclease is used as a recognitionrestriction endonuclease, a restriction endonuclease other than an EcoRIrestriction endonuclease (e.g., a HindIII restriction endonuclease) isused as an amplifying restriction endonuclease. Examples of restrictionendonucleases that can be used as amplifying restriction endonucleasesinclude, without limitation, EcoRI, EcoRII, BamHI, HindIII, TaqI, NotI,HinfI, Sau3A, PovII, SmaI, HaeIII, HgaI, AluI, EcoRV, EcoP15I, KpnI,PstI, SacI, SalI, ScaI, SphI, StuI, XbaI, AarI, BanII, BseGI, BspPI,CfrI, EcoNI, Hsp92II, NlaIV, RsaI, TaiI, AasI, BbsI, BseLI, BspTI, ClaI,EcoO109I, I-PpoI, NmuCI, RsrII, TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI,KasI, Acc65I, BbvCI, BseMI, BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI,AccB7I, BbvI, BseMII, BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI,BceAI, BseNI, BsrGI, CspI, Esp3I, KspAI, NsiI, SapI, and TauIrestriction endonucleases. Any number of molecules of the sameamplifying restriction endonuclease can be attached to one probe nucleicacid molecule. For example, a single probe nucleic acid molecule cancontain one, two, three, four, five, or more EcoRI amplifyingrestriction endonuclease molecules. In some cases, a single probenucleic acid molecule can contain two or more (e.g., two, three, four,five, or more) different types of amplifying restriction endonucleases.For example, a single probe nucleic acid molecule can contain threeEcoRI amplifying restriction endonuclease molecules and two BanIIamplifying restriction endonuclease molecules.

Any appropriate method can be used to attach an amplifying restrictionendonuclease to a nucleic acid component of the probe nucleic acid. Insome cases, an amplifying restriction endonuclease can be attached by anionic or covalent attachment. For example, covalent bonds such as amidebonds, disulfide bonds, and thioether bonds, or bonds formed bycrosslinking agents can be used. In some cases, a non-covalent linkagecan be used. The attachment can be a direct attachment or an indirectattachment. For example, a linker can be used to attach an amplifyingrestriction endonuclease to a nucleic acid component of the probenucleic acid. In some cases, nucleic acid can include a thiolmodification, and a restriction endonuclease can be conjugated to thethiol-containing nucleic acid based on succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) using techniquessimilar to those described elsewhere (Dill et al., Biosensors andBioelectronics, 20:736-742 (2004)). In some cases, a biotinylatednucleic acid and a streptavidin-containing restriction endonuclease canbe attached to one another via a biotin-streptavidin interaction. Arestriction endonuclease can be conjugated with streptavidin using, forexample, sulfosuccinimidyl6-(3′-[2-pyridyldithio]-propionamido)hexanoate. An amplifyingrestriction endonuclease can be attached at any location of a nucleicacid component of the probe nucleic acid. For example, an amplifyingrestriction endonuclease can be attached at an end (e.g., a 5′ end or 3′end) of a nucleic acid component, in the middle of a nucleic acidcomponent, or at any position along the length of a nucleic acidcomponent.

Signal expansion nucleic acid (e.g., first signal expansion nucleic acidand second signal expansion nucleic acid) and reporter nucleic aciddescribed herein typically include at least one double-stranded DNAsection that includes an amplifying restriction endonuclease cut site(e.g., an initial amplifying restriction endonuclease cut site, asecondary amplifying restriction endonuclease cut site, or a tertiaryamplifying restriction endonuclease cut site). The other portions of thesignal expansion nucleic acid or reporter nucleic acid can include DNA,RNA, or other molecules. For example, reporter nucleic acid can includebiotin such that the reporter nucleic acid can be attached to astreptavidin-coated solid support. In some cases, one or both strands ofthe double-stranded section of the signal expansion nucleic acid or thereporter nucleic acid that contains an amplifying restrictionendonuclease cut site can be RNA or a nucleic acid analog (e.g., apeptide nucleic acid (PNA)) provided that such a double-stranded sectionis capable of being cleaved by the amplifying restriction endonuclease.Examples of restriction endonucleases that can be used as amplifyingrestriction endonucleases to cleave a DNA:RNA hybrid section of signalexpansion nucleic acid or reporter nucleic acid include, withoutlimitation, HhaI, AluI, TaqI, HaeIII, EcoRI, HindII, SalI, and MspIrestriction endonucleases.

Signal expansion nucleic acid or reporter nucleic acid described hereincan be any length provided that the double-stranded section thatcontains the amplifying restriction endonuclease cut site is capable ofbeing cleaved by the amplifying restriction endonuclease. In general,the double-stranded section of signal expansion nucleic acid or reporternucleic acid can be between about 10 and about 500 or more nucleotides(e.g., between about 10 and about 400 nucleotides, between about 10 andabout 300 nucleotides, between about 10 and about 200 nucleotides,between about 10 and about 100 nucleotides, between about 10 and about50 nucleotides, between about 10 and about 25 nucleotides, between about20 and about 500 nucleotides, between about 30 and about 500nucleotides, between about 40 and about 500 nucleotides, between about50 and about 500 nucleotides, between about 15 and about 50 nucleotides,between about 15 and about 25 nucleotides, between about 20 and about 50nucleotides, or between about 18 and about 25 nucleotides, between about20 and about 60 nucleotides, between about 25 and about 55 nucleotides,between about 30 and about 50 nucleotides, between about 35 and about 45nucleotides, or between about 38 and about 42 nucleotides) in length. Insome cases, the double-stranded section of signal expansion nucleic acidor reporter nucleic acid can be between 5 and 50 nucleotides in length.The amplifying restriction endonuclease cut site of the signal expansionnucleic acid or the reporter nucleic acid can be located at any positionalone the double-stranded section. For example, the amplifyingrestriction endonuclease cut site can be towards the 5′ end, towards the'3 end, or near the center of the double-stranded section of the signalexpansion nucleic acid or the reporter nucleic acid. In general, theoverall length of signal expansion nucleic acid or reporter nucleic aciddescribed herein can be between about 10 and about 2500 or morenucleotides (e.g., between about 10 and about 2000 nucleotides, betweenabout 10 and about 1000 nucleotides, between about 10 and about 500nucleotides, between about 10 and about 400 nucleotides, between about10 and about 300 nucleotides, between about 10 and about 200nucleotides, between about 10 and about 100 nucleotides, between about10 and about 50 nucleotides, between about 10 and about 25 nucleotides,between about 20 and about 500 nucleotides, between about 30 and about500 nucleotides, between about 40 and about 500 nucleotides, betweenabout 50 and about 500 nucleotides, between about 75 and about 500nucleotides, between about 100 and about 500 nucleotides, between about150 and about 500 nucleotides, between about 15 and about 50nucleotides, between about 15 and about 25 nucleotides, between about 20and about 50 nucleotides, between about 18 and about 25 nucleotides,between about 20 and about 60 nucleotides, between about 25 and about 55nucleotides, between about 30 and about 50 nucleotides, between about 35and about 45 nucleotides, or between about 38 and about 42 nucleotides)in length.

The amplifying restriction endonuclease cut site of signal expansionnucleic acid or reporter nucleic acid described herein can be a cut siteof any type of restriction endonuclease. In addition, any type ofrestriction endonuclease can be used as an amplifying restrictionendonuclease to cleave signal expansion nucleic acid or reporter nucleicacid. Examples of restriction endonucleases that can be used asamplifying restriction endonucleases include, without limitation, EcoRI,EcoRII, BamHI, HindIII, TaqI, NotI, HinfI, Sau3A, PovII, SmaI, HaeIII,HgaI, AluI, EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI, SphI, StuI,XbaI, AarI, BanII, BseGI, BspPI, CfrI, EcoNI, Hsp92II, NlaIV, RsaI,TaiI, AasI, BbsI, BseJI, BspTI, ClaI, EcoO109I, I-PpoI, NmuCI, RsrII,TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI, KasI, Acc65I, BbvCI, BseMI,BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI, AccB7I, BbvI, BseMII, BsrFI,Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI, BceAI, BseNI, BsrGI, CspI,Esp3I, KspAI, NsiI, SapI, and TauI restriction endonucleases.

In general, signal expansion nucleic acid or reporter nucleic acid canbe designed to have a double-stranded section that contains a singleamplifying restriction endonuclease cut site. In some cases, signalexpansion nucleic acid or reporter nucleic acid provided herein can bedesigned to have a double-stranded section that contains more than one(e.g., two, three, four, five, six, seven, eight, nine, ten, or more)amplifying restriction endonuclease cut site. When more than oneamplifying restriction endonuclease cut site is used, the multipleamplifying restriction endonuclease cut sites can be cut sites for thesame restriction endonuclease or cut sites for different restrictionendonucleases. For example, reporter nucleic acid can be designed tohave a double-stranded section that contains one initial amplifyingrestriction endonuclease cut site for an EcoRI initial amplifyingrestriction endonuclease and one secondary amplifying restrictionendonuclease cut site for an XbaI secondary amplifying restrictionendonuclease.

Any appropriate method can be used to obtain the nucleic acid componentof signal expansion nucleic acid or reporter nucleic acid. For example,common molecular cloning and chemical nucleic acid synthesis techniquescan be used to obtain the nucleic acid component of signal expansionnucleic acid or reporter nucleic acid. In some cases, the nucleic acidcomponent of signal expansion nucleic acid or reporter nucleic acid canbe synthesized using commercially available automated oligonucleotidesynthesizers such as those available from Applied Biosystems (FosterCity, Calif.). In some cases, signal expansion nucleic acid or reporternucleic acid can be synthesized de novo using any of a number ofprocedures widely available in the art. Examples of such methods ofsynthesis include, without limitation, the β-cyanoethyl phosphoramiditemethod (Beaucage et al., Tet. Let., 22:1859-1862 (1981)) and thenucleoside H-phosphonate method (Garegg et al., Tet. Let., 27:4051-4054(1986); Froehler et al., Nucl. Acid Res., 14:5399-5407 (1986); Garegg etal., Tet. Let., 27:4055-4058 (1986); and Gaffney et al., Tet. Let.,29:2619-2622 (1988)). These methods can be performed by a variety ofcommercially-available automated oligonucleotide synthesizers. In somecases, recombinant nucleic acid techniques such as PCR and those thatinclude using restriction enzyme digestion and ligation of existingnucleic acid sequences (e.g., genomic DNA or cDNA) can be used to obtainthe nucleic acid component of signal expansion nucleic acid or reporternucleic acid.

Signal expansion nucleic acid or reporter nucleic acid described hereincan be attached to a solid support. Examples of solid supports include,without limitation, a well of a microtiter plate (e.g., a 96-wellmicrotiter plate or ELISA plate), beads (e.g., magnetic, glass, plastic,or gold-coated beads), slides (e.g., glass or gold-coated slides),micro- or nano-particles (e.g., carbon nanotubes), platinum solidsupports, palladium solid supports, and a surface of a chamber orchannel within a microfluidic device. In some cases, a solid support canbe a silicon oxide-based solid support, a plastic polymer-based solidsupport (e.g., a nylon, nitrocellulose, or polyvinylidene fluoride-basedsolid support) or a biopolymer-based (e.g., a cross-linked dextran orcellulose-based solid support) solid support.

Signal expansion nucleic acid or reporter nucleic acid can be directlyor indirectly attached to a solid support. For example, biotin can be acomponent of signal expansion nucleic acid or reporter nucleic acid, andthe signal expansion nucleic acid or the reporter nucleic acidcontaining biotin can be indirectly attached to a solid support that iscoated with streptavidin via a biotin-streptavidin interaction. In somecases, signal expansion nucleic acid or reporter nucleic acid can beattached to a solid support via a covalent or non-covalent interaction.For example, signal expansion nucleic acid or reporter nucleic acid canbe covalently attached to magnetic beads as described elsewhere(Albretsen et al., Anal. Biochem., 189(1):40-50 (1990)).

Signal expansion nucleic acid can be designed to contain any type ofrestriction endonuclease as an amplifying restriction endonuclease(e.g., an initial amplifying restriction endonuclease, a secondaryamplifying restriction endonuclease, or a tertiary amplifyingrestriction endonuclease). In general, an amplifying restrictionendonuclease of signal expansion nucleic acid is typically a differentrestriction endonuclease than the restriction endonuclease that is usedas a recognition restriction endonuclease. For example, when an EcoRIrestriction endonuclease is used as a recognition restrictionendonuclease, a restriction endonuclease other than an EcoRI restrictionendonuclease (e.g., a HeaIII restriction endonuclease) is used as anamplifying restriction endonuclease. Examples of restrictionendonucleases that can be used as amplifying restriction endonucleasesinclude, without limitation, EcoRI, EcoRII, BamHI, HindIII, TaqI, NotI,HinfI, Sau3A, PovII, SmaI, HaeIII, HgaI, AluI, EcoRV, EcoP15I, KpnI,PstI, SacI, SalI, ScaI, SphI, StuI, XbaI, AarI, BanII, BseGI, BspPI,CfrI, EcoNI, Hsp92II, NlaIV, RsaI, TaiI, AasI, BbsI, BseLI, BspTI, ClaI,EcoO109I, I-PpoI, NmuCI, RsrII, TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI,KasI, Acc65I, BbvCI, BseMI, BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI,AccB7I, BbvI, BseMII, BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI,BceAI, BseNI, BsrGI, CspI, Esp3I, KspAI, NsiI, SapI, and TauIrestriction endonucleases. Any number of molecules of the sameamplifying restriction endonuclease can be attached to one signalexpansion nucleic acid molecule. For example, a single signal expansionnucleic acid molecule can contain one, two, three, four, five, or moreEcoRI amplifying restriction endonuclease molecules. In some cases, asingle signal expansion nucleic acid molecule can contain two or more(e.g., two, three, four, five, or more) different types of amplifyingrestriction endonucleases. For example, a single signal expansionnucleic acid molecule can contain three BanII amplifying restrictionendonuclease molecules and two SacII amplifying restriction endonucleasemolecules.

Reporter nucleic acid can be designed to contain a label to aid in thedetection of cleaved reporter nucleic acid. In some cases, signalexpansion nucleic acid can be designed to contain a label. In suchcases, signal expansion nucleic acid containing a label can be used inaddition to reporter nucleic acid or in place of reporter nucleic acidto detect target nucleic acid. Examples of labels that can be acomponent of reporter nucleic acid or signal expansion nucleic acidinclude, without limitation, fluorescent labels (with or without the useof quenchers), dyes, antibodies, radioactive material, enzymes (e.g.,horse radish peroxidase, alkaline phosphatase, laccase, galactosidase,or luciferase), redox labels (e.g., ferrocene redox labels), metallicparticles (e.g., gold nanoparticles), and green fluorescentprotein-based labels. In some cases, for a redox label, such asferrocene, the detector can be an electrode for amperometric assay ofredox molecules. For example, if the redox label is present in a reducedform of ferrocene, then the electrode at high electrode potential canprovide an oxidation of the reduced form of ferrocene, therebyconverting it to an oxidized form of ferrocene. The generated currentcan be proportional to the concentration of ferrocene label in thesolution.

In one embodiment, reporter nucleic acid or signal expansion nucleicacid can contain a fluorescent label and a quencher such that cleavedreporter nucleic acid provides a fluorescent signal and uncleavedreporter nucleic acid does not provide a fluorescent signal. In somecases, the reporter nucleic acid or signal expansion nucleic acid cancontain a label (e.g., a fluorescent label or an enzyme such as horseradish peroxidase) and can be attached to a solid support (e.g., a wellof a microtiter plate). For example, the reporter nucleic acid or signalexpansion nucleic acid can be attached to a solid support such thatcleavage at the amplifying restriction endonuclease cut site by theamplifying restriction endonuclease releases a portion of the reporternucleic acid or the signal expansion nucleic acid that contains thelabel. The resulting reaction mixture can be collected and assessed forthe presence, absence, or amount of released portions of the reporternucleic acid or signal expansion nucleic acid using the label. Forexample, the released portions of the reporter nucleic acid or thesignal expansion nucleic acid, if present, can be transferred from onewell of a microtiter plate (e.g., a 96-well plate) that contained thereporter nucleic acid or the signal expansion nucleic acid to anotherwell of a microtiter plate, where the transferred material can beassessed for a signal from the label. Any number of molecules of a labelcan be attached to one reporter nucleic acid molecule or one signalexpansion nucleic acid molecule. For example, a reporter nucleic acidmolecule or a single signal expansion nucleic acid molecule can containone, two, three, four, five, or more fluorescent molecules.

Any appropriate method can be used to attach a label to a nucleic acidcomponent of reporter nucleic acid or signal expansion nucleic acid. Insome cases, a label can be attached by an ionic or covalent attachment.For example, covalent bonds such as amide bonds, disulfide bonds, andthioether bonds, or bonds formed by crosslinking agents can be used. Insome cases, a non-covalent linkage can be used. The attachment can be adirect attachment or an indirect attachment. For example, a linker canbe used to attach a label to a nucleic acid component of reporternucleic acid or signal expansion nucleic acid. In some cases, nucleicacid can include a thiol modification, and a label can be conjugated tothe thiol-containing nucleic acid based on succinimidyl4-[N-maleimidomethyl]cyclo-hexane-1-carboxylate (SMCC) using techniquessimilar to those described elsewhere (Dill et al., Biosensors andBioelectronics, 20:736-742 (2004)). In some cases, a biotinylatednucleic acid and a streptavidin-containing label can be attached to oneanother via a biotin-streptavidin interaction. A label can be conjugatedwith streptavidin using, for example, sulfosuccinimidyl6-(3′-[2-pyridyldithio]-propionamido)hexanoate. A label can be attachedat any location of a nucleic acid component of reporter nucleic acid orsignal expansion nucleic acid. For example, a label can be attached atan end (e.g., a 5′ end or 3′ end) of a nucleic acid component, in themiddle of a nucleic acid component, or at any position along the lengthof a nucleic acid component of reporter nucleic acid or signal expansionnucleic acid.

As described herein, the methods and materials provided herein can beused to detect target nucleic acid containing a genetic or epigeneticelement in any type of sample (e.g., a biological sample). For example,a blood sample or cheek swab sample can be collected from a mammal andassessed for target nucleic acid to determine if the mammal has one ormore genetic or epigenetic elements of interest. Once obtained, a sampleto be assessed can be processed to obtain nucleic acid. For example, anucleic acid extraction can be performed on a blood sample to obtain asample that is enriched for nucleic acid. In some cases, a sample can beheated or treated with a cell lysis agent to release nucleic acid fromcells present in the sample.

As described herein, a sample (e.g., a biological sample) can beassessed for the presence, absence, or amount of target nucleic acid(e.g., target nucleic acid containing a genetic or epigenetic element)using an enzymatic amplification cascade of restriction endonucleasesdescribed herein without using a nucleic acid amplification technique(e.g., a PCR-based nucleic acid technique). Assessing samples (e.g.,biological samples) for the presence, absence, or amount of targetnucleic acid using an enzymatic amplification cascade of restrictionendonucleases described herein without using a nucleic acidamplification technique can allow patients as well as medical,laboratory, or veterinarian personnel (e.g., clinicians, physicians,physician's assistants, laboratory technicians, research scientists, andveterinarians) to test for one or more genetic or epigenetic elementswithout the need for potentially expensive thermal cycling devices andpotentially time consuming thermal cycling techniques. In some cases,the methods and materials provided herein can be used in combinationwith a PCR-based nucleic acid technique. For example, a PCR-basednucleic acid technique can be performed to amplify nucleic acid (e.g., atarget nucleic acid containing a genetic or epigenetic element) presentwithin a biological sample, and the resulting amplification material canbe assessed using an enzymatic amplification cascade of restrictionendonucleases described herein to detect the presence, absence, oramount of a particular nucleic acid (e.g., a target nucleic acid agenetic or epigenetic element). In some cases, a limited PCR-basednucleic acid technique can be performed to amplify a target nucleic acidto a point where the amount of amplified target nucleic acid isincreased only slightly over the amount of target nucleic acidoriginally present within the biological sample. For example, a two totwelve cycle PCR technique (e.g., a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12 cycle PCR technique) can be performed to slightly increase the amountof amplified target nucleic acid as compared to the amount ofunamplified target nucleic acid originally present within the biologicalsample. Such limited PCR-based nucleic acid techniques, when used incombination with an enzymatic amplification cascade of restrictionendonucleases described herein, can allow medical, laboratory, orveterinarian personnel to test organisms with a potentially increasedlevel of sensitivity and/or specificity without the potentially lengthytime involved in thermal cycling techniques that include a greaternumber of cycles. This increased level of sensitivity and/or specificitycan be over the high level of sensitivity and specificity of acomparable testing procedure that includes an enzymatic amplificationcascade of restriction endonucleases described herein without thelimited PCR-based nucleic acid technique. In some cases, the PCR-basednucleic acid technique can be performed to amplify a target nucleic acidto a point where the amount of amplified target nucleic acid is easilydetectable (e.g., visually detectable using gel electrophoresis andethidium bromide staining) For example, a 15 or more cycle PCR technique(e.g., a 20 cycle PCR technique) can be performed to produce at least ngamounts (e.g., greater than 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, or more)of amplified nucleic acid. Such PCR-based nucleic acid techniques, whenused in combination with an enzymatic amplification cascade ofrestriction endonucleases described herein, can allow medical,laboratory, or veterinarian personnel to test organisms with apotentially increased level of sensitivity and/or specificity. Thisincreased level of sensitivity and/or specificity can be over the highlevel of sensitivity and specificity of a comparable testing procedurethat includes an enzymatic amplification cascade of restrictionendonucleases described herein without the PCR-based nucleic acidtechnique.

In some cases, a sample (e.g. a biological sample) can be obtained andsubjected to a culturing technique. For example, a cell sample can beobtained and cultured with medium (e.g., enrichment medium) to enrichthe sample such that the number of cells present in the sample canincrease. Examples of enrichment media include, without limitation,Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM),Iscove's Modified Dulbecco's Media (IMDM), and AIM V® Medium. In somecases, the culture medium can contain a nutrient (e.g. serum such asfetal calf serum), ingredient, or drug that prevents certain cells fromdividing while allowing other cells to divide. In some cases, theculturing technique can include incubating a sample at an appropriatetemperature (e.g. between 15° C. and 45° C., between 20° C. and 45° C.,between 25° C. and 45° C., between 30° C. and 45° C., between 30° C. and40° C., between 35° C. and 45° C., or between 35° C. and 40° C.) for anappropriate period of time (e.g., between about 0.5 hours and 48 hours,between about 0.5 hours and 36 hours, between about 0.5 hours and 24hours, between about 0.5 hours and 12 hours, between about 0.5 hours and8 hours, between about 0.5 hours and 6 hours, between about 0.5 hoursand 5 hours, between about 0.5 hours and 4 hours, between about 0.5hours and 3 hours, between about 0.5 hours and 2 hours, between about 1hour and 4 hours, or between about 2 hours and 4 hours). For example, asample can be obtained and cultured in tissue culture medium at 37° C.for 24-48 hours. Examples of tissue culture techniques that can be usedas described herein include, without limitation, those describedelsewhere (Animal Cell Culture: A Practical Approach, 3rd edition, J.Masters, ed., Oxford University Press, 2000, 336 pp).

In some cases, a sample, obtained and subjected to a culturing techniqueor not, can be processed, for example, to remove non-nucleic acidmaterial, to disrupt cell membranes to release nucleic acid, and/or tocollect or extract nucleic acid, such that nucleic acid of the sample,if present within the sample, is available for hybridization to probenucleic acid. For example, a blood or cheek swab sample can be treatedwith a lysis buffer and subjected to nucleic acid extraction such that amajor component of the sample is nucleic acid. In some cases, a samplecan be homogenized and treated to disrupt cells that are present in thesample. For example, a blood sample can be subjected to high speedmechanical homogenization with glass/silica/zirconium/stainless steelbeads, can be subjected to high temperature (e.g., boiling orautoclaving), can be subjected to chemical lysis with detergents and/orsurfactants (e.g., sodium dodecyl sulfate, cetyltrimethylammoniumbromide, or sodium lauroyl sarcosin), can be subjected to one or morefreeze-thaw cycles using, e.g., liquid nitrogen or dry ice, can besubjected to sonication, or can be subjected to combinations thereof.The resulting sample can be subjected to a standard nucleic acidextraction technique such as those described elsewhere (e.g., Sambrookand Russell, (2001) Molecular Cloning: A Laboratory Manual, ThirdEdition, Cold Spring Harbor Press) or a nucleic acid extractiontechnique that includes the use of magnetic beads or selectiveDNA-binding membranes (see, e.g., QIAGEN DNeasy® Blood & Tissue Kit, orMo Bio PowerFood™ Microbial DNA Isolation Kit). For example, the bloodsample can be contacted with magnetic beads that bind nucleic acid, thebeads can be removed, and bound nucleic acid can be eluted into anappropriate buffer to form a processed sample for further analysis usingthe methods and materials provide herein. Such a process can be carriedout using a variety of kits including, without limitation, QiagenBioSprint 96 One-For-All Vet Kit (a rapid and economical automatedpurification of viral nucleic acid and/or bacterial nucleic acid fromsamples based on magnetic beads) and Chemicell geneMAG-PCR cleanup kit.In some cases, a sample (e.g., a blood sample or body fluid sample) canbe subjected to DNA isolation using Qiagen QIAcard FTA Spots or QiagenQIAamp UltraSens Virus Kits.

In some cases, a sample can be processed in a manner designed tofragment any nucleic acid present within the sample. For example,genomic or large pieces of nucleic acid present within a sample can besubjected to a sonication technique, nebulization technique, and/orrestriction digestion with a restriction endonuclease such as DpnII orCviJI to generate nucleic acid fragments. Such fragmentation can beperformed using restriction endonucleases that are different from thoseused as recognition or amplifying restriction endonucleases to assessthe sample as described herein.

In some cases, the sample can be treated such that any double-strandednucleic acid present within the sample is separated. For example, abiological sample can be heated and then snap-cooled or can be subjectedto chemical (e.g., sodium hydroxide) denaturation. In some cases, whenthe sample is subjected to a PCR-based technique, certain primer orreaction modifications can be used to generate preferentiallysingle-stranded product. For example, unidirectional DNA polymerasereactions can be performed with a single specific primer. In some cases,the strands of nucleic acid can be separated, and the strand of interestcan be enrichment using specific biotinylated primers andstreptavidin-conjugated magnetic beads. In some cases, selectivedigestion of one of the strands can be accomplished using lambdaexonucleases.

As described herein, a sample (e.g., a biological sample) can besubjected to a nucleic acid amplification technique. For example, atissue sample containing extracted nucleic acid can be subjected to aquick PCR-based amplification of one or more specific targets (e.g., 1hour, end-point PCR) or to a whole genome amplification technique (e.g.,Qiagen REPLI-g Screening Kit for high-throughput manual or automatedwhole genome amplification).

Once obtained, a sample to be assessed, whether subjected to a PCR-basednucleic acid technique or not, can be contacted with a probe nucleicacid as described herein. This contacting step can be carried out forany period of time and at any temperature that allows target nucleicacid to hybridize with probe nucleic acid. For example, this step can beperformed between 10 seconds and 24 hours (e.g., between 30 seconds and12 hours, between 30 seconds and 8 hours, between 30 seconds and 4hours, between 30 seconds and 2 hours, between 30 seconds and 1 hour,between 1 minute and 24 hours, between 1 minute and 12 hours, between 1minute and 8 hours, between 1 minute and 4 hours, between 1 minute and 2hours, between 1 minute and 1 hour, between 5 minutes and 1 hour,between 10 minutes and 1 hour, between 15 minutes and 1 hour, or between30 minutes and 1 hour). The initial temperature can be between 15° C.and 100° C. (e.g., between 23° C. and 98° C., between 23° C. and 90° C.,between 23° C. and 85° C., between 23° C. and 75° C., between 23° C. and65° C., between 23° C. and 55° C., between 23° C. and 45° C., between23° C. and 35° C., between 30° C. and 95° C., between 30° C. and 85° C.,between 30° C. and 75° C., between 30° C. and 65° C., between 30° C. and55° C., between 30° C. and 45° C., between 20° C. and 40° C., between20° C. and 30° C., and between 25° C. and 35° C.). The temperatureduring this contacting step can remain constant or can be increased ordecreased. For example, the initial temperature can be between about 40°C. and about 85° C., and then the temperature can be allowed to decreaseto room temperature over a period of about 30 seconds to about 30minutes (e.g., between about 30 seconds and about 15 minutes, betweenabout 30 seconds and about 10 minutes, between about 1 minute and about30 minutes, between about 1 minute and about 15 minutes, or betweenabout 1 minute and about 5 minutes).

Contact of the sample (e.g., a biological sample to be tested) withprobe nucleic acid can occur in the presence of the recognitionrestriction endonucleases, or a separate step of adding the recognitionrestriction endonucleases to the reaction can be performed. Therecognition restriction endonuclease step can be carried out for anyperiod of time and at any temperature that allows the recognitionrestriction endonuclease to cleave recognition restriction endonucleasecut sites formed by the hybridization of target nucleic acid to theprobe nucleic acid. For example, this step can be performed between onesecond and 24 hours (e.g., between one second and 30 minutes, betweenone second and one hour, between five seconds and one hour, between 30seconds and 24 hours, between 30 seconds and 12 hours, between 30seconds and 8 hours, between 30 seconds and 4 hours, between 30 secondsand 2 hours, between 30 seconds and 1 hour, between 1 minute and 24hours, between 1 minute and 12 hours, between 1 minute and 8 hours,between 1 minute and 4 hours, between 1 minute and 2 hours, between 1minute and 1 hour, between 5 minutes and 1 hour, between 10 minutes and1 hour, between 15 minutes and 1 hour, or between 30 minutes and 1hour). The temperature can be between 15° C. and 75° C. (e.g., between15° C. and 75° C., between 15° C. and 65° C., between 15° C. and 55° C.,between 15° C. and 45° C., between 15° C. and 35° C., between 15° C. and30° C., between 23° C. and 55° C., between 23° C. and 45° C., between30° C. and 65° C., between 30° C. and 55° C., between 30° C. and 45° C.,between 30° C. and 40° C., between 35° C. and 40° C., and between 36° C.and 38° C.). Any appropriate concentration of recognition restrictionendonuclease can be used. For example, between about 0.001 units and1000 units (e.g., between about 0.001 units and 750 units, between about0.001 units and 500 units, between about 0.001 units and 250 units,between about 0.001 units and 200 units, between about 0.001 units and150 units, between about 0.001 units and 100 units, between about 0.001units and 50 units, between about 0.001 units and 25 units, betweenabout 0.001 units and 10 units, between about 0.001 units and 1 unit,between about 0.001 units and 0.1 units, between about 0.01 units and1000 units, between about 0.1 units and 1000 units, between about 1 unitand 1000 units, between about 10 units and 1000 units, between about 50units and 1000 units, between about 0.5 units and 100 units, or betweenabout 1 unit and 100 units) of restriction endonuclease can be used.Other restriction endonuclease reaction conditions such as saltconditions can be used according to the manufacturer's instructions.

When one step of a method provided herein is completed, the resultingreaction product containing cleaved nucleic acid can be used in the nextstep. For example, cleaved nucleic acid of a reaction product can beremoved from uncleaved nucleic acid and used in the next step of themethod. For example, when probe nucleic acid is attached to a solidsupport, the released portions of probe nucleic acid that contain anamplifying restriction endonuclease can be collected and placed incontact with reporter nucleic acid or signal expansion nucleic acid asdescribed herein. The resulting reaction products of a particular stepcan be manually or automatically (e.g., robotically) transferred to alocation containing nucleic acid for the next step (e.g., reporternucleic acid or signal expansion nucleic acid), which nucleic acid canbe attached or not attached to a solid support. In some cases, onereaction of a method described herein can be carried out at one location(e.g., a chamber) of a microfluidic device or blister package device,and the reaction products that are generated can be moved to anotherlocation (e.g., another chamber) that contains nucleic acid for the nextstep (e.g., reporter nucleic acid or signal expansion nucleic acid) viaa channel. In some cases, cleaved nucleic acid of a reaction product canbe used in the next step of the method by removing the uncleaved nucleicacid from the reaction product. For example, when magnetic beads areused as a solid support, a magnetic force can be used to remove themagnetic beads and any attached uncleaved nucleic acid from the reactionproduct. In some cases, two or more reactions of a method providedherein can be carried out at one location (e.g., a single well of amicrotiter plate or a single chamber of a microfluidic device). Forexample, a single compartment can have one region that containsimmobilized probe nucleic acid and another region that containsimmobilized reporter nucleic acid provided that the amplifyingrestriction endonuclease of the immobilized probe nucleic acid is notcapable of cleaving the amplifying restriction endonuclease cut site ofthe reporter nucleic acid unless target nucleic acid hybridizes to theprobe nucleic acid and the recognition restriction endonuclease cleavesthe probe nucleic acid, thereby releasing a portion of the probe nucleicacid that contains the amplifying restriction endonuclease so that it iscapable of cleaving the reporter nucleic acid. In another example, asingle compartment can have one region that contains immobilized probenucleic acid, other regions that contain immobilized signal expansionnucleic acid (e.g., one region that contains a first signal expansionnucleic acid and another region that contains a second signal expansionnucleic acid), and another region that contains immobilized reporternucleic acid provided that the amplifying restriction endonucleases ofimmobilized probe nucleic acid and signal expansion nucleic acid are notcapable of cleaving their intended amplifying restriction endonucleasecut sites until they are released as described herein. Such singlecompartments can be made using partitions or sub-compartments within thesingle compartment. For example, a sample to be tested can be placedinto a single well of a microtiter plate that contains probe nucleicacid, recognition restriction endonucleases, first and second signalexpansion nucleic acid, and reporter nucleic acid such that cleavedreporter nucleic acid and/or signal expansion nucleic acid is producedas described herein when target nucleic acid is present in the samplebeing tested and little or no cleaved reporter nucleic acid and/orsignal expansion nucleic acid is produced when target nucleic acid isnot present in the sample being tested.

Any appropriate method can be used to detect cleaved reporter nucleicacid and/or signal expansion nucleic acid to determine the presence,absence, or amount of target nucleic acid in a sample, which canindicate the presence, absence, or amount of a target genetic orepigenetic element. For example, size separation techniques can be usedto assess reaction products for cleaved reporter nucleic acid and/orsignal expansion nucleic acid. Examples of such size separationtechniques include, without limitation, gel electrophoresis andcapillary electrophoresis techniques. In some cases, a melt curveanalysis can be performed to assess reaction products for cleavedreporter nucleic acid and/or signal expansion nucleic acid. As describedherein, a label can be used to aid in the detection of cleaved nucleicacid (e.g., reporter nucleic acid and/or signal expansion nucleic acid).Examples of labels that can be used include, without limitation,fluorescent labels (with or without the use of quenchers), dyes,antibodies, radioactive material, enzymes (e.g., horse radishperoxidase, alkaline phosphatase, laccase, galactosidase, orluciferase), redox labels (e.g., ferrocene redox labels), metallicparticles (e.g., gold nanoparticles), and green fluorescent proteinbased labels. For example, the release of fluorescently labeled portionsof reporter nucleic acid and/or signal expansion nucleic acid from asolid support can be assessed using common fluorescent label detectors.In some cases, cleaved reporter nucleic acid and/or signal expansionnucleic acid can be detected electrochemically. For electrochemicaldetection, the reporter nucleic acid and/or signal expansion nucleicacid can include a ferrocene redox label. Reporter nucleic acid and/orsignal expansion nucleic acid containing ferrocene can be obtained bycoupling ferrocene carboxylic acid with an amino-modifiedoligonucleotide using the carbodiimide reaction in the presence of anexcess of ferrocene carboxylic acid. In one embodiment, for a redoxlabel, such as ferrocene, the detector can be an electrode foramperometric assay of redox molecules. For example, if the redox labelis present in a reduced form of ferrocene, then the electrode at highelectrode potential can provide an oxidation of the reduced form offerrocene, thereby converting it to an oxidized form of ferrocene. Thegenerated current can be proportional to the concentration of ferrocenelabel in the solution.

The methods and materials provided herein can be used to assess one ormore samples for target nucleic acid in real-time. For example, afluorescent label/quencher system or an electrochemical redox labelsystem can be used to detect cleavage of reporter nucleic acid and/orsignal expansion nucleic acid in real time.

The methods and materials provided herein can be used to assess one ormore samples (e.g., two, three, four, five, six, seven, eight, nine,ten, 20, 50, 100, 500, 1000, or more) for a single type of targetnucleic acid. For example, 100s of tissue samples (e.g., tissue biopsysamples) can be assessed for target nucleic acid containing a particulargenetic or epigenetic element. In some case, the methods and materialsprovided herein can be used in a multiplex manner to assess one or moresamples for more than one (e.g., two, three, four, five, six, seven,eight, nine, ten, 20, 50, 100, 500, 1000, or more) type of targetnucleic acid. For example, target nucleic acid for ten differentsequences (e.g., ten different SNP sequences) can be used to design tendifferent probe nucleic acid molecules. In these cases, each probenucleic acid can be used in a separate series of reactions within thesame device (e.g., microtiter plate or microfluidic device), and thesame label can be used for the reporter nucleic acid for each probenucleic acid. In addition, in some cases, the same amplifyingrestriction endonuclease can be used for each probe nucleic acid, andthe same reporter nucleic acid can be used for each reaction series. Insome cases, when multiple different probe nucleic acid molecules areused in the same reaction series, a different reporter nucleic acidhaving different labels can be used to correspond to each probe nucleicacid such that the detected signals can indicate which of the ten targetnucleic acids are being detected.

This document also provides kits for performing the methods describedherein. For example, a kit provided herein can include probe nucleicacid with or without being attached to a solid support and/or reporternucleic acid with or without being attached to a solid support. In somecases, such a kit can include a recognition restriction endonuclease,first signal expansion nucleic acid, second signal expansion nucleicacid, or a combination thereof. In some cases, a kit can be configuredinto a microfluidic device that allows for the movement of probe nucleicacid, first signal expansion nucleic acid, second signal expansionnucleic acid, reporter nucleic acid, or recognition restrictionendonucleases (or any combination thereof) as well as a cleaved portionof any such nucleic acid in a manner that allows a detection methodprovided herein to be carried out with or without the nucleic acid beingattached to a solid support. For example, a kit provided herein can be amicrofluidic device capable of receiving a sample and contacting thatsample with probe nucleic acid. The probe nucleic acid can be designedto include a length of nucleotides followed by the sequencecomplementary to the target nucleic acid, which can create a recognitionrestriction endonuclease cut site, followed by an amplifying restrictionendonuclease. The distance from the recognition restriction endonucleasecut site to the amplifying restriction endonuclease can be relativelyshort (e.g., 100, 50, 25, 10, or less nucleotides), while the distancefrom the recognition restriction endonuclease cut site to the beginningof the length of nucleotides can be relatively long (e.g., 50, 100, 150,200, 500, 1000, 2000, or more). In such cases, cleavage of the probenucleic acid at the recognition restriction endonuclease cut site canresult in a relatively small portion that contains the amplifyingrestriction endonuclease and is capable of travelling faster than thelarger uncleaved probe nucleic acid. This difference can allow thecleaved portion containing the amplifying restriction endonuclease toreach an area of the microfluidic device containing signal expansionnucleic acid or reporter nucleic acid so that the next reaction can becarried out without the presence of uncleaved probe nucleic acid. Insome cases, after the smaller portion containing the amplifyingrestriction endonuclease enters the area containing signal expansionnucleic acid or reporter nucleic acid, a valve can be used to preventthe larger uncleaved probe nucleic acid from entering. In some cases, afilter can be used to limit the ability of larger uncleaved probenucleic acid from proceeding to the next reaction location. Similarapproaches can be used during other steps of a method provided herein toseparate cleaved nucleic acid from uncleaved nucleic acid.

In some cases, a kit provided herein can be a portable or self-containeddevice, packet, vessel, or container that can be used, for example, inpoint of care applications. For example, such a kit can be configured toallow a patient or physician's assistant to insert a sample foranalysis. In some cases, a kit can be designed for use in a home settingor any other setting. Once inserted, the sample can be heated (e.g.,heated to about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90,95, or more ° C.) and/or cooled by a heating or cooling mechanismlocated within the kit. For example, an exothermic or endothermicchemical reaction can be initiated within the kit to increase, decrease,or maintain the temperature. Such exothermic or endothermic chemicalreactions can be carried out within the kit without being in fluidcommunication with the reactions of the target nucleic acid detectionmethod. An iron oxidation reaction is an example of an exothermicchemical reaction that can be used to heat a kit provided herein. Anendothermic chemical reaction that can be used to cool a kit providedherein can be a reaction that includes the use of ammonium chloride andwater, potassium chloride and water, or sodium carbonate and ethanoicacid. In general, when detecting DNA target nucleic acid, the kit can bedesigned to generate, if needed, enough heat to denature double strandedDNA present within the sample. The kit also can be designed to generateappropriate heating and cooling temperatures to carry out each step of adetection method provided herein. In some cases, a kit provided hereincan include a temperature indicator (e.g., color indicator orthermometer) to allows a user to assess temperature.

In some cases, a kit can be designed to provide a user with a “yes” or“no” indication about the presence of target nucleic acid within atested sample. For example, a label having the ability to generate achange in pH can be used, and a visual indicator (e.g., a pH-based colorindicator) can be used to inform the user of the presence of targetnucleic acid based on a change in pH.

In some cases, a point of care or home use device can be designed tocarry out the reactions described herein. For example, point of care orhome use device can be designed to include a series of adjacentchambers. In a relatively simple configuration, for example, a first“sample” chamber can be configured for sample insertion, and can containreagents (e.g., in dry or liquid form) to effect generation of singlestranded nucleic acid fragments. A second “recognition” chamber can beconfigured to receive single stranded nucleic acid fragments from thefirst chamber, and can contain probe nucleic acid and recognitionrestriction endonuclease (e.g., in dry or liquid form). A third“amplification” chamber can be configured to receive cleaved portions ofprobe nucleic acid from the second chamber, and can contain reporternucleic acid (e.g., in dry or liquid form). A fourth “detection” chambercan be configured to receive cleaved portions of marker nucleic acidfrom the third chamber, and can contain a reagent (e.g., in dry orliquid form) that serves as an indicator of whether or not targetnucleic acid was present in the sample. It is noted that one or moreadditional “signal expansion” chambers can be present between the“recognition” chamber and the “amplification” chamber.

In some cases, a point of care or home use device can be configured suchthe chambers are separated from each other by membranes that can providecontrolled passage of reaction materials. For example, chambers can beseparated by membranes that are subject to degradation by particularreagents or solutions. In such cases, a reaction can be confined to aparticular chamber until the membrane separating it from the adjacentchamber degrades, permitting passage of reaction components therebetween.

In some cases, a point of care or home use device can be adapted forautomatic transfer of the reaction mixture between chambers. Forexample, insertion of a sample into the first chamber can trigger areaction or provide a reagent that gradually degrades the membraneseparating the first chamber from the second chamber. Movement of all ora portion of the reaction mixture into the second chamber can in turnprovide a reagent or trigger a reaction that gradually degrades themembrane separating the second chamber from the third chamber. Forexample, if the sample reaction mixture in the first chamber is anaqueous solution, the reagents in the second chamber are dry, and themembrane in the second chamber is degraded by water, movement of theaqueous reaction mixture into the second chamber can trigger degradationof the membrane therein.

In some cases, a point of care or home use device can be adapted forautomatic controlled flow transfer of reaction mixture between chambers.For example, insertion of a sample into the first chamber can trigger areaction or provide a reagent that allows controlled flow movement ofthe sample through absorption media. Movement of all or a portion of thereaction mixture into the second chamber can in turn provide a reagentor trigger a reaction that allows controlled flow movement of the samplethrough absorption media to a third chamber. In such cases, a reactioncan be confined to a particular chamber until the media separating itfrom the adjacent chamber absorbs and permits passage of reactioncomponents there between.

In some cases, a point of care or home use device can be adapted forautomatic controlled flow transfer of reaction mixture between chambers.For example, insertion of a sample into the first chamber can trigger areaction or provide a reagent that allows controlled capillary flowmovement of the sample through micro-fluidic channels. Movement of allor a portion of the reaction mixture into the second chamber can in turnprovide a reagent or trigger a reaction that allows controlled flowmovement of the sample through micro-fluidic channels to a thirdchamber. In such cases, a reaction can be confined to a particularchamber until the microfluidic channel permits passage of reactioncomponents there between.

In some cases, a point of care or home use device can be adapted forautomatic controlled flow transfer of reaction mixture without chambers.For example, insertion of a sample into the device can trigger areaction or provide a reagent that allows controlled capillary flowmovement of the sample through microfluidic channels. Movement of all ora portion of the reaction mixture in the microfluidic channel cantrigger a reaction that allows reagents to enter the reaction mixture ina continuous flow-through manner with no specific chamber for areaction. In such cases, a reaction does not need to be confined to aparticular section of the microfluidic channel.

In some cases, transfer of a reaction mixture from one chamber to thenext can be controlled by a user. An exemplary user-controlled,pen-style point of care or home use device is depicted in FIG. 7. Device300 can include sample collector 310 and reaction unit 320. Samplecollector 310 can have cap 312 with screw threads 314, shaft 316, andswabber 318. Swabber 318 can be smooth or rough, and in some cases canhave bristles (e.g., smooth or rough bristles) or a matted texture tofacilitate sample collection from, for example, the inside cheek,throat, or skin of an individual to be tested.

Reaction unit 320 can include tube 322, open end 324 reversibly closedby safety cap 326, and closed end 328. Open end 324 can have internalscrew threads, and cap 326 can have external screw threads 329. Screwthreads 329 of safety cap 326, as well as screw threads 314 of samplecollector cap 312, can be adapted to mate with the internal screwthreads at open end 324, such that either safety cap 326 or samplecollector 310 can be screwed into open end 324.

Tube 322 can contain several chambers, such as lysing and isolationchamber 330, recognition and amplification chamber 360, and detectionchamber 390. As described herein, the chambers can be separated from oneanother to prevent premature mixing of reaction components. Tube 322 andthe chambers contained therein can be made from, for example, clearplastic (e.g., polycarbonate, acrylic, nylon, or PVC). Tube 322 also cancontain first and second safety bands 340 and 370, and first and secondspring returns 350 and 380.

Lysing and isolation chamber 330 can be positioned proximal to open end324. Lysing and isolation chamber 330 can have proximal end 332, distalend 334, proximal membrane 336, distal membrane 337, and reactioncompletion indicator 338. Proximal membrane 336 can be located adjacentto proximal end 332, and distal membrane 337 can be located adjacent todistal end 334. Membranes 336 and 337 can be made from, for example,synthetic rubber, natural latex rubber, or silicone. Chamber 330 cancontain reagents for lysing cells as well as reagents for cleaving anddenaturing cellular nucleic acids. Reaction completion indicator 338 canbe, for example, a built in timer or stop watch, a built in pHindicator, a built in color change reagent, or a conductivity probe, andcan indicate when cell lysis and nucleic acid sample generation aresufficient to proceed to the next step.

First safety band 340 can be positioned distal to lysing and isolationchamber 330 within tube 322, and first spring return 350 can bepositioned distal to first safety band 340. First safety band 340 canbe, for example, connected to a tab or strap, and can be moved orremoved from reaction unit 320 by pulling on the tab or strap. Firstspring return 350 can be made from a shape memory material that can becompressed and then automatically return to or toward its originalconfiguration.

The safety band 340 can be attached to the tube as a secured ring thatcan be, for example, over molded as a soft rubber component or insertedas a spring like split ring component. The safety band 340 can lock theposition of the lysing and isolation tube chamber 340, preventing linearsliding of the lysing and isolation chamber 330 to that of therecognition and amplification chamber 360. Upon removal of safety band340, the user can actuate linear movement of the entire device 300 byholding the proximal end firm and pressing the distal closed end 328such that both distal chambers recognition and amplification 360 anddetection chamber 390 are moved toward the lysing and isolation chamber330. The needle and sample collector 362 can pierce membrane 337 andenter the lysing and isolation chamber 330. The user can release a firmhold on the assembly and spring return 350 can draw the sample intorecognition and amplification chamber 360. After completion of thereaction, the user can remove safety band 370, and the user can actuatelinear movement of the assembly by holding the recognition andamplification chamber 360 firm and pressing the distal closed end 328such that the detection chamber 390 moves toward the recognition andamplification chamber 360. The needle and sample collector 392 canpierce membrane 366. The user can release the firm hold on the assembly,and spring return 380 can draw the sample into detection chamber 390.

Recognition and amplification chamber 360 can be positioned distal tofirst spring return 350. Chamber 360 can have proximal end 361, which inturn can have piercing needle and sample collector 362, distal end 364,membrane 366, and reaction completion indicator 368. Recognition andamplification chamber 360 can contain, for example, probe nucleic acidand reporter nucleic acid and restriction endonucleases for use inenzymatic amplification cascades as described herein. Piercing needleand sample collector 362 can have a pointed, beveled, or barbed tip. Inaddition, the interior of piercing needle and sample collector 362 canbe in fluid communication with the interior of recognition andamplification chamber 360, such that a nucleic acid test sample can becollected from lysing and isolation chamber 330 and transferred torecognition and amplification chamber 360 via collector 362. Membrane364 can be located adjacent to distal end 364, and can be made from, forexample, synthetic rubber, natural latex rubber, or silicone. Reactioncompletion indicator 368 can be, for example, a built in timer or stopwatch, a built in pH indicator, a built in color change reagent, or aconductivity probe, and can indicate when cell lysis and nucleic acidsample generation are sufficient to proceed to the next step.

Second safety band 370 can be positioned distal to recognition andamplification chamber 360 within tube 322, and second spring return 380can be positioned distal to second safety band 370. Second safety band370 can be, for example, connected to a tab or strap, and can be movedor removed from reaction unit 320 by pulling on the tab or strap. Secondspring return 380 can be made from a shape memory material (e.g., springsteel, plastic, or rubber) that can be compressed and then automaticallyreturn to or toward its original configuration.

Detection chamber 390 can be positioned distal to second spring return380, adjacent to closed end 328 of tube 322. Detection chamber 390 canhave proximal end 391, which in turn can have piercing needle and samplecollector 392, and distal end 394. Piercing needle and sample collector392 can have a pointed, beveled, or barbed tip. In addition, theinterior of piercing needle and sample collector 392 can be in fluidcommunication with the interior of detection chamber 390, such that areaction sample can be collected from recognition and amplificationchamber 360 and transferred to detection chamber 390 via collector 392.Detection chamber 390 can contain a substrate for an enzyme marker suchas a substrate for horseradish peroxidase (HRP) (e.g., ABTS, TMB, OPD)or alkaline phosphatase (AP) (e.g., PNNP).

Sample collector 310 and reaction unit 320 can be packaged together andsold as a kit. In use, the sample collector 310 can be removed from thepackage, and a swab can be obtained from, for example, a subject's body.Cap 326 can be removed from open end 324 of tube 322, and samplecollector 310 can be screwed into open end 324 such that all or aportion of swabber 318 extends through proximal membrane 336 and intothe interior of lysing and isolation chamber 330. The sample can bemixed (e.g., by shaking), and the lysing and nucleic acid preparationcan proceed for a particular length of time, or until reactioncompletion indicator 338 indicates that the user can proceed to the nextreaction step.

When the nucleic acid sample is ready, the user can remove first safetyband 350 from reaction unit 320, and can actuate reaction unit 320 suchthat piercing needle and sample collector 362 moves proximally topenetrate distal membrane 337 of lysing and isolation chamber 330,collects a sample from chamber 330, and, by virtue of first springreturn 350, moves distally to its original position. The sample canagain be mixed, and the recognition and amplification steps can proceedfor a particular length of time, or until reaction completion indicator368 indicates that the user can proceed to the next reaction step.

When the reaction sample is ready, the user can remove second safetyband 380 from reaction unit 320, and can actuate reaction unit 320 suchthat piercing needle and sample collector 392 moves proximally topenetrate membrane 366 of recognition and amplification chamber 360,collects a sample from chamber 360, and, by virtue of second springreturn 380, moves distally to its original position. The sample canagain be mixed, and marker released during the amplification step can bedetected (e.g., colorimetrically or fluorescently). In some cases, theouter surface of tube 322 can have a color code printed thereon, so auser can compare the color of detection chamber 390 with the color codeto determine whether or not the tested sample contains target nucleicacid.

Device 300 can have any suitable dimensions. For example, the size ofdevice 300 can approximate that of a pen or a marker, which can make itparticularly convenient to transport. In some cases, device 300 can havea diameter at its widest point of about 0.25 to about 2 cm (e.g., 0.25,0.3, 0.4, 0.5, 0.6, 0.75, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, or 2 cm), and a length of about 5 cm to about 200 cm(e.g., 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, or 200 cm).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Formation and Cleavage of Target-Probe Hybrids

An oligonucleotide probe (5′-thiol-GGT AGT GCG AAA TGC CAT TGC TAG TTGTTT-biotin-3′; SEQ ID NO:2) that was modified with a thiol group at the5′ end and a biotin molecule at the 3′ end was conjugated to horseradishperoxidase (HRP). Conjugation was performed using the SMCC reagentaccording to a technique modified from Dill et al. (Biosensors andBioelectronics, 20:736-742 (2004)). The HRP conjugate solution wasincubated with a streptavidin-coated ELISA plate to immobilize theHRP-oligonucleotide probe to the surface via a biotin-streptavidininteraction. The ELISA plate was then incubated with differentconcentrations of a target oligonucleotide (5′-AAA CAA CTA GCA ATG GCATTT-3′; SEQ ID NO:3). The target oligonucleotide sequence wasreverse-complementary to the probe sequence to form a double-strandedhybrid molecule. After washing, the plate was incubated in a solutioncontaining the restriction endonuclease BfaI. BfaI specificallyrecognizes the sequence 5′-CTAG-3′ and cleaves the double-stranded,target-probe hybrids to release the HRP-oligonucleotide into thereaction solution. After a two-hour incubation at 37° C., the reactionsolution was transferred to a new ELISA plate. The cleavedHRP-oligonucleotide was contacted to 3,3′,5,5′-tetramethyl benzidine(TMB) to form a colored reaction product.

When the restriction endonuclease BfaI was added in excess to thereaction mixture, a clear direct dependence between the amount ofreleased HRP-probe and the concentration of oligonucleotide target wasobserved (FIG. 6A). The detectable target concentration wasapproximately 1 nM. This detection limit was obtained by directmeasurement without any secondary signal amplification. The addition ofa restriction endonuclease signal amplification cascade as describedherein can further improve the detection limit by several orders ofmagnitude.

When the HRP-oligonucleotide probes were pre-incubated with an excess oftarget oligonucleotide (500 nM), the amount of cleavedHRP-oligonucleotide probe was limited by the amount of recognitionrestriction endonuclease BfaI (FIG. 6B). Taken together, these datademonstrate that recognition restriction endonucleases can be used toinitiate the restriction endonuclease cascades described herein.

Example 2 Detecting Target Nucleic Acid Using Probe Nucleic Acid andReporter Nucleic Acid

A target nucleic acid is selected. Once selected, target nucleic acid isanalyzed using a common genetic database such as GenBank® and/or acomputer-based sequence analysis program to identify a portion of thetarget nucleic acid that contains a cut site for a restrictionendonuclease. Probe nucleic acid is designed to be complementary to atleast a portion of target nucleic acid that contains a cut site. Oncedesigned and obtained by standard oligonucleotide synthesis methods,probe nucleic acid is conjugated to an amplifying restrictionendonuclease and immobilized to the surface of a first well of amicrotiter plate. A sample to be tested is incubated in the first well.If target nucleic acid is present in the sample, at least a portion ofthe target nucleic acid hybridizes to the probe nucleic acid, andthereby forms a recognition restriction endonuclease cut site. Therecognition restriction endonuclease is added to the first well havingthe sample and probe nucleic acid. The microtiter plate is incubated at37° C. for an appropriate length of time for the cleavage reaction toproceed.

Upon cleavage of probe nucleic acid by the recognition restrictionendonuclease, the reaction solution containing the released portion ofthe probe nucleic acid is transferred into a second well. The secondwell contains reporter nucleic acid that is immobilized to the surfaceand contains at least one double-stranded portion having an amplifyingrestriction endonuclease cut site. Reporter nucleic acid also has afluorescent label. Upon transfer to the second chamber, the amplifyingrestriction endonuclease bound to the released portion of the probenucleic acid contacts the reporter nucleic acid. The amplifyingrestriction endonuclease cleaves reporter nucleic acid at thedouble-stranded amplifying restriction endonuclease cut site to form atleast two portions. The liberated portion of the reporter nucleic acidhaving the fluorescent label is moved to a third microtiter plate well,and a standard fluorescent reader is used to measure any fluorescentsignal.

A standard curve of known amounts of target nucleic acid is used toquantify the amount of target nucleic acid in the tested sample.

Example 3 Detecting Target Nucleic Acid Using Probe Nucleic Acid, FirstSignal Expansion Nucleic Acid, Second Signal Expansion Nucleic Acid, andReporter Nucleic Acid

Once selected, target nucleic acid is analyzed using a common geneticdatabase such as GenBank® and/or a computer-based sequence analysisprogram to identify a portion of target nucleic acid that contains a cutsite for a restriction endonuclease. Probe nucleic acid is designedbased on the desired target nucleic acid as described herein. Standardoligonucleotide synthesis methods are used to make the probe nucleicacid, which is then conjugated to an initial amplifying restrictionendonuclease and immobilized to the surface of a first well of amicrotiter plate. A sample to be tested for the target nucleic acid isincubated in the first well. If target nucleic acid is present in thesample, at least a portion of target nucleic acid hybridizes to probenucleic acid and thereby forms a recognition restriction endonucleasecut site. Recognition restriction endonuclease is added to the firstwell having the sample and probe nucleic acid. The microtiter plate isincubated at 37° C. for an appropriate length of time for the cleavagereaction to proceed.

After cleavage of the probe nucleic acid:target nucleic acid hybrid bythe recognition restriction endonuclease, the reaction solutioncontaining the free portion of probe nucleic acid is transferred toanother well that includes first signal expansion nucleic acid andsecond signal expansion nucleic acid. The first signal expansion nucleicacid and second signal expansion nucleic acid creates a positivefeedback loop that causes an exponential acceleration of release ofinitial amplifying restriction enzymes. The reaction product from thiswell is transferred to another well containing reporter nucleic acid,and cleavage of the reporter nucleic acid is used to determine thepresence, absence, or amount of target nucleic acid in the sample. Astandard curve of known amounts of target nucleic acid is used toquantify the amount of target nucleic acid in the tested sample.

Example 4 Detecting Methylated Cyclin D2 Promoter in Circulating Bloodof Breast Cancer Patients

The presence or absence methylated cyclin D2 promoter in circulatingblood of breast cancer patients can be indicative of breast cancerlesions (Rykova et al., Ann. N.Y. Acad. Sci., 1137:232-235 (2008)). Thepresence or absence of methylated cyclin D2 promoter is determined intotal circulating DNA (cirDNA) from the blood that can include cell-freeand cell-surface-bound DNA fractions isolated as described elsewhere(e.g., Sunami et al., Methods Mol. Biol., 507:349-356 (2009)). Thepresence or absence methylated cyclin D2 promoter is detected using anenzymatic amplification cascade. The gene for cyclin D2 (CCDN2,G1/S-specific cyclin-D2; Ensembl ID: ENSG00000118971) is located in theforward strand of human Chromosome 12 at position 4,382,902-4,414,521.Its promoter is composed of at least 7 fragments, and the longest 700 bpfragment (sequence ID ENSR00000172023; chromosome 12 positions4386022-4386721) was analyzed using the Ensembl (“http” colon, slash,slash “uswest” dot “ensemble” dot “org” slash “index” dot “html”)genetic database and CLC DNA Workbench software to identify a portion oftarget sequence with a cut site for the MspI/HpaII restrictionendonucleases, which cleave at the 4 bp nucleotide sequence 5′-CCGG-3′.A 40 nt probe nucleic acid(5′-GTTTATTGGGGTGCTTTACCCCGGCTGTACACAGAAAGCC-3′ (SEQ ID NO:4)) wasdesigned to be complementary to nucleotides 520 to 559 of the selectedtarget nucleic acid (chromosome 12 positions 4386542-4386581).

Once designed and obtained by standard oligonucleotide synthesismethods, probe nucleic acid is conjugated to an amplifying restrictionendonuclease and immobilized to the surface of two wells of a microtiterplate. A sample of circulating DNA to be tested is obtained from patientblood or body fluids, and added to the two wells. If the cyclin D2promoter sequence is present in circulating DNA, it will bind to theprobe in both wells thereby forming a CCGG restriction site for the MspIrecognition restriction endonuclease, which is methylation insensitive,and the HpaII recognition restriction endonuclease, which is methylationsensitive. MspI and HpaII are either added or present in the first andsecond wells, respectively, and they are allowed to cleave any formedrecognition restriction endonuclease cut sites by incubating themicrotiter plate at 37° C. for an appropriate length of time (e.g., 1minute to 2 hours) for the cleavage reaction to proceed.

After cleavage of the probe nucleic acid:target nucleic acid hybrid byMspI and HpaII, the reaction solutions in the first and second wells aretransferred to third and fourth wells, respectively, both containingreporter nucleic acid that is immobilized to the surface and that has atleast one double-stranded portion having an amplifying restrictionendonuclease NcoI cut site. The reporter nucleic acid can be adouble-stranded nucleic acid having a first strand (e.g.,5′-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3′ (SEQ ID NO:5)) and a secondstrand (e.g., 5′-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3′ (SEQ ID NO:6)).The reporter nucleic acid also has a fluorescent label. In some cases,first signal expansion nucleic acid and second signal expansion nucleicacid are used prior to the reporter nucleic acid step to increase thelevel of target nucleic acid detection. The first signal expansionnucleic acid and second signal expansion nucleic acid can includelabels, in which case they can be used together with reporter nucleicacid or in place of reporter nucleic acid.

After transferring the reaction mixture to the third and fourth wells,the amplifying restriction endonucleases of the released portions ofprobe nucleic acid contact reporter nucleic acid, and the microtiterplate is incubated at an appropriate temperature (e.g., at 37° C.) foran appropriate length of time (e.g., 1 minute to 2 hours) for thecleavage reaction to proceed. The amplifying restriction endonucleasescleave reporter nucleic acid at the double-stranded amplifyingrestriction endonuclease cut site to form at least two portions. Thereaction solutions of the third and fourth wells are transferred tofifth and six wells, respectively, for fluorescence detection using afluorescent microtiter plate reader. The fluorescent signal in the fifthwell (corresponding to the MspI recognition restrictionendonuclease-treated well) is indicative of total amount of the cyclinD1 promoter in the circulating blood. The fluorescent signal in thesixth well (corresponding to the HpaII recognition restrictionendonuclease-treated well) is indicative of the amount of unmethylatedcyclin D1 promoter in the circulating blood. If the latter signal issmaller than the former signal then at least a part of the correspondingcyclin D2 promoter DNA is methylated. The proportion of methylatedpromoter can be calculated as a difference between the signal in thefifth well minus the signal in the sixth well.

Example 5 Detecting Methylated RASSF1A Promoter in Circulating Blood ofHepatocellular Carcinoma and Lung Cancer Patients

The presence or absence methylated RASSF1A promoter in circulating bloodof hepatocellular carcinoma and lung cancer patients is indicative oftumor growth and disease progression (Di Gioia et al., BMC Cancer, 6:89(2006); Fischer et al., Lung Cancer, 56:115-123 (2007); and Allen Chanet al., Clin. Chem., 10:1373 (2008)). The presence or absence ofmethylated RASSF1A promoter is determined in total circulating DNA(cirDNA) from the blood that can include cell-free andcell-surface-bound DNA fractions isolated as described elsewhere (e.g.,Sunami et al., Methods Mol. Biol., 507:349-356 (2009)).

The presence or absence methylated RASSF1A promoter is detected using anenzymatic amplification cascade. The gene for RASSF1A (Ras associationdomain-containing protein 1; Ensembl ID: ENSG00000068028) is located inthe reverse strand of Chromosome 3 at positions 50,367,217-50,378,411.Its promoter is composed of at least three fragments, and the longest1,702 bp fragment (sequence ID ENSR00000059407, Chromosome3:50369843-50371544) was analyzed using the Ensembl(http://uswest.ensembl.org/index.html) genetic database and CLC DNAWorkbench software to identify a portion of target sequence with cutsites for two restriction endonucleases, PstI and SmaI. PstI cleaves atthe 6 bp nucleotide sequence 5′-CTGCAG-3′, and a 40 nt probe nucleicacid (5′-AGTCCGAGTCCTCTTGGCTGC-AGTAGCCACTGCTCGTCGT -3′ (SEQ ID NO:7))was designed to be complementary to nucleotides 503 to 542 of theselected target nucleic acid (Chromosome 3:50370346-50370385). SmaIcleaves at the 6 bp nucleotide sequence 5′-CCCGGG-3′, and a 40 nt probenucleic acid (5′-GTGTCAGTGTGCGCGTGCGCCCGGGCCAGAGCCGCGCCGC-3′ (SEQ IDNO:8)) was designed to be complementary to nucleotides 746 to 785 of theselected target nucleic acid (Chromosome 3:50370589-50370628). The PstIcut site is not methylated, and the SmaI cut site is a CpG island thatcan be methylated in cancer cells, and its methylation blocks cleavageby SmaI.

Once designed and obtained by standard oligonucleotide synthesismethods, probe nucleic acids are conjugated to an amplifying restrictionendonuclease and immobilized to the surface of two wells of a microtiterplate. A sample of circulating DNA to be tested is obtained from patientblood or body fluids, and added to the two wells. If the RASSF1Apromoter sequence is present in circulating DNA, it will bind to theprobe nucleic acid in both wells thereby forming restriction sites forPstI and for SmaI in the first and second wells, respectively. PstI andSmaI are either added or present in the first and second wells,respectively, and they are allowed to cleave any formed recognitionrestriction endonuclease cut sites by incubating the microtiter plate at37° C. for an appropriate length of time (e.g., 1 minute to 2 hours) forthe cleavage reaction to proceed.

After cleavage of the probe nucleic acid:target nucleic acid hybrids byPstI or SmaI, the reaction solutions in the first and second wells aretransferred to third and fourth wells, respectively, both containingreporter nucleic acid that is immobilized to the surface and that has atleast one double-stranded portion having an amplifying restrictionendonuclease NcoI cut site. The reporter nucleic acid can be adouble-stranded nucleic acid having a first strand (e.g.,5′-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3′ (SEQ ID NO:5)) and a secondstrand (e.g., 5′-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3′ (SEQ ID NO:6)).The reporter nucleic acid also has a fluorescent label. In some cases,first signal expansion nucleic acid and second signal expansion nucleicacid are used prior to the reporter nucleic acid step to increase thelevel of target nucleic acid detection. The first signal expansionnucleic acid and second signal expansion nucleic acid can includelabels, in which case they can be used together with reporter nucleicacid or in place of reporter nucleic acid.

After transferring the reaction mixture to the third and fourthchambers, the amplifying restriction endonucleases of the releasedportions of probe nucleic acid contact reporter nucleic acid, and themicrotiter plate is incubated at an appropriate temperature (e.g., at37° C.) for an appropriate length of time (e.g., 1 minute to 2 hours)for the cleavage reaction to proceed. The amplifying restrictionendonucleases cleave reporter nucleic acid at the double-strandedamplifying restriction endonuclease cut site to form at least twoportions. The reaction solutions of the third and fourth wells aretransferred to fifth and six wells, respectively, for fluorescencedetection using a fluorescent microtiter plate reader. The fluorescentsignal in the fifth well (corresponding to the PstI recognitionrestriction endonuclease-treated well) is indicative of total amount ofthe RASSF1A promoter in the circulating blood. The fluorescent signal inthe sixth well (corresponding to the SmaI recognition restrictionendonuclease-treated well) is indicative of the amount of unmethylatedRASSF1A promoter in the circulating blood. If the latter signal issmaller than the former signal then at least a part of the correspondingRASSF1A promoter DNA is methylated. The proportion of methylatedpromoter can be calculated as the difference between the signal in thefifth well minus the signal in the sixth well.

Example 6 Assessing Alleles of the Thiopurine S-Methyltransferase GeneBased on a Sequence that Creates/Destroys a Restriction EndonucleaseSite

Thiopurine S-methyltransferase (EC 2.1.1.67) enzyme (TPMT) is adrug-metabolizing enzyme that catalyzes the S-methylation of thiopurinedrugs such as 6-mecaptopurine and azathioprine that are used to treatchildhood leukemia, autoimmune diseases, and transplant recipients (Wanget al., Proc. Natl. Acad. Sci. USA, 102(26):9394-9399 (2005)). Thesedrugs can have potentially life-threatening drug-induced toxicity,depending on the levels of the TPMT enzyme in patient's tissues. Largeindividual variations in levels of TPMT activity are regulated primarilyby common genetic polymorphisms with several most common alleles. Someof these alleles can result in a virtual lack of TPMT enzyme activity,and correspondingly, patients homozygous for these alleles can suffersevere, life-threatening toxicity when treated with standard doses ofthiopurines.

The known SNP variants of the TPMT gene (Ensemble gene IDENSG00000137364; positioned on Chromosome 6: 18,128,542-18,155,305reverse strand) were analyzed using the Ensembl (“http” colon, slash,slash, “uswest” dot “ensemble” dot “org” slash “index” dot “html”)genetic database and CLC DNA Workbench to determine whether these SNPscreate or destroy restriction sites. One of these SNPs (Ensembl IDrs72552739; chromosome 6 position 18143901, C to A substitution causingpremature stop codon and truncated 98-amino acid truncated polypeptide)was selected since this C/A substitution destroyed an ApoI restrictionsite 5′-AAATTC-3′ present in un-mutated DNA (chromosome 6 positions18143896-18143901). Another restriction site for BfuI, 5′-GTATCC-3′ wasfound to be present in both mutated and un-mutated target DNA(chromosome 6 positions 18143904-18143909). Two probes (P1 and P2) weredesigned to be complementary to nucleotides 18143882-18143922 of theselected target nucleic acid(5′-TTCTGCTCTGTAAAAAATTcTTGTATCCCAAGTTCACTGAT-3′ (P1; SEQ ID NO:9) and5′-TTCTGCTCTGTAAAAAATTaTTGTATCCCAAGTTCACTGAT-3′ (P2; SEQ ID NO:10)), onecorresponding to the un-mutated (P1), and another to mutated DNA (P2),respectively (the SNP positions are shown with lowercase letters).

Once designed and obtained by standard oligonucleotide synthesismethods, probe nucleic acid P1 is conjugated to an amplifyingrestriction endonuclease (NcoI) and immobilized to the surface of twowells of a microtiter plate. A sample of genomic DNA to be tested isobtained from a patient's blood (with or without PCR amplification andsingle-stranded target preparation), and added to the two wells. If thetarget nucleic acid for P1 is present in the genomic DNA, it will bindto P1 in both wells thereby forming restriction sites for recognitionrestrictases ApoI and BfuI. ApoI is added to the first well, and BfuI isadded to the second well, and they are allowed to cleave any formedrecognition restriction endonuclease cut sites by incubating themicrotiter plate at 37° C. for an appropriate length of time (e.g., 1minute to 2 hours) for the cleavage reaction to proceed.

After cleavage of the P1: target nucleic acid hybrids by ApoI or BfuI,the reaction solutions in the first and second wells are transferred tothird and fourth wells, respectively, both containing reporter nucleicacid that is immobilized to the surface and that has at least onedouble-stranded portion having an amplifying restriction endonucleaseNcoI cut site. The reporter nucleic acid can be a double-strandednucleic acid having a first strand (e.g.,5′-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3′ (SEQ ID NO:5)) and a secondstrand (e.g., 5′-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3′ (SEQ ID NO:6)).The reporter nucleic acid also has a fluorescent label. In some cases,first signal expansion nucleic acid and second signal expansion nucleicacid are used prior to the reporter nucleic acid step to increase thelevel of target nucleic acid detection. The first signal expansionnucleic acid and second signal expansion nucleic acid can includelabels, in which case they can be used together with reporter nucleicacid or in place of reporter nucleic acid.

After transferring the reaction mixture to the third and fourthchambers, the amplifying restriction endonucleases of the releasedportions of P1 contact reporter nucleic acid, and the microtiter plateis incubated at an appropriate temperature (e.g., at 37° C.) for anappropriate length of time (e.g., 1 minute to 2 hours) for the cleavagereaction to proceed. The amplifying restriction endonucleases cleavereporter nucleic acid at the double-stranded amplifying restrictionendonuclease cut site to form at least two portions. The reactionsolutions of the third and fourth wells are transferred to fifth and sixwells, respectively, for fluorescence detection using a fluorescentmicrotiter plate reader. The fluorescent signal in the fifth well isindicative of the amount of un-mutated ApoI cleaved TPMT allelic variantin the sample. The TPMT allelic variant containing the C/A SNP, ifpresent, is not cleaved and doesn't contribute to the signal in thefifth well. The fluorescent signal in the sixth wells is indicative oftotal amount of the target TPMT nucleic acid in the sample, both mutatedand un-mutated. Thus, the allelic composition of the patient's genotypein terms of the corresponding SNP can be evaluated from the ratio ofun-mutated TPMT allele to total amount of the TPMT nucleic acid (signalin the fifth well versus signal in the sixth well). A ratio ofapproximately 0.5 is indicative of heterozygosity. A ratio ofapproximately 1 is indicative of homozygosity of the un-mutated allele,and a ratio close to zero is indicative of homozygosity of the mutatedallele.

Example 7 Assessing Alleles of the Thiopurine S-Methyltransferase GeneBased on a Sequence that does not Appear to Create/Destroy a RestrictionEndonuclease Site

An allele of the TPMT gene (Ensemble gene ID ENSG00000137364; positionedon Chromosome 6: 18,128,542-18,155,305 reverse strand) was selected todesign an enzymatic amplification cascade of restriction endonucleasesusing a recognition restriction endonuclease that has separaterecognition and cleavage sites (FokI) since the SNP of this allele doesnot appear to create or destroy a restriction site. The SNP (Ensembl IDrs1800460; chromosome 6 position 18139228, C to T substitution causingnon-synonymous substitution in the codon 154) is one of the most commonvariant alleles (Wang et al., Proc. Natl. Acad. Sci. USA,102(26):9394-9399 (2005)). The corresponding nucleic acid sequence wasanalyzed using the Ensembl (“http” colon, slash, slash, “uswest” dot“ensemble” dot “org” slash “index” dot “html”) genetic database and CLCDNA Workbench to select the SNP site (position 18139228) and flankingsequences, 8 nucleotides upstream (5′ direction, 18139220-18139227), and31 nucleotides downstream (3′ direction, 18139229-18139259). Thesesequences are used to design the single stranded parts of two probenucleic acids (P1 and P2), one corresponding to the non-mutated geneticelement (P1, 5′-ATAGAGGAcCATTAGTTGCCATCAATCCAGGTGAT-CGCAA-3′; (SEQ IDNO:11)), and one corresponding to the mutated genetic element (P2,5′-ATAGAGGAtCATTAGTTGCCATCAATCCAGGTGATCGCAA-3′ (SEQ ID NO:12)). Thecommon double-stranded part of the P1 and P2 probe nucleic acidscontained a 15-bp spacer followed by the FokI recognition site:5′-CATTGCGCGCCTAGTGGATG-3′ (SEQ ID NO:13) as shown in FIG. 13.

Once designed and obtained by standard oligonucleotide synthesismethods, the probe nucleic acids are conjugated to an amplifyingrestriction endonuclease (NcoI) and immobilized to the surface of twowells of a microtiter plate. The first and second wells thus contain P1for the un-mutated DNA and P2 for the mutated DNA, respectively. Asample of genomic DNA to be tested is obtained from a patient's blood(with or without PCR amplification and single-stranded targetpreparation), and added to the two wells. If the target nucleic acid ispresent in the genomic DNA, it can hybridize to the probe nucleic acidin the wells and create a FokI cleavage site with its correspondingprobe nucleic acid. The protruding ends of target nucleic acid areremoved using blunting by adding T4 DNA polymerase, and then the bluntednucleic acid is ligated to the adjacent available strand of the probenucleic acid using E. coli DNA ligase by incubating the microtiter plateat 20 to 37° C. for an appropriate length of time (e.g., 1 minute to 2hours) for the enzymatic reactions to proceed. FokI is added to bothfirst and second wells, and is allowed to cleave any formed cleavagesites by incubating the microtiter plate at 37° C. for an appropriatelength of time (e.g., 1 minute to 2 hours) for the cleavage reaction toproceed. FokI is only able to cleave the perfect match between the probenucleic acid and target nucleic acid, and not a single nucleotidemismatch at the cleavage site. After cleavage of the probe nucleicacid:target nucleic acid hybrids by FokI, the reaction solutions in thefirst and second wells are transferred to third and fourth wells,respectively, both containing reporter nucleic acid that is immobilizedto the surface and that has at least one double-stranded portion havingan amplifying restriction endonuclease NcoI cut site. The reporternucleic acid can be a double-stranded nucleic acid having a first strand(e.g., 5′-CATTGCTAGTTGTTTCCATGGGGTA-GTGCGAAATGC-3′ (SEQ ID NO:5)) and asecond strand (e.g., 5′-GCATTTCGCAC-TACCCCATGGAAACAACTAGCAATG-3′ (SEQ IDNO:6)). The reporter nucleic acid also has a fluorescent label. In somecases, first signal expansion nucleic acid and second signal expansionnucleic acid are used prior to the reporter nucleic acid step toincrease the level of target nucleic acid detection. The first signalexpansion nucleic acid and second signal expansion nucleic acid caninclude labels, in which case they can be used together with reporternucleic acid or in place of reporter nucleic acid.

After transferring the reaction mixture to the third and fourthchambers, the amplifying restriction endonucleases of the releasedportions of probe nucleic acid contact reporter nucleic acid, and themicrotiter plate is incubated at an appropriate temperature (e.g., at37° C.) for an appropriate length of time (e.g., 1 minute to 2 hours)for the cleavage reaction to proceed. The amplifying restrictionendonucleases cleave reporter nucleic acid at the double-strandedamplifying restriction endonuclease cut site to form at least twoportions. The reaction solutions of the third and fourth wells aretransferred to fifth and six wells, respectively, for fluorescencedetection using a fluorescent microtiter plate reader. The fluorescentsignal in the fifth wells is indicative of the amount of the un-mutatedTPMT target nucleic acid in the sample. The fluorescent signal in thesixth well is indicative of the amount of mutated TPMT target nucleicacid in the sample. Thus, the allelic composition of the patient'sgenotype in terms of the corresponding SNP can be evaluated from theratio of un-mutated TPMT allele to mutated TPMT allele (signal in thefifth well versus signal in the sixth well). The ratio of approximately1 is indicative of heterozygosity. If the signal in the fifth wellgreatly exceeding the one in the sixth well, then the results areindicative of homozygosity for the un-mutated allele, and the oppositeis indicative of homozygosity for the mutated allele.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for assessing an organism for a genetic or epigeneticelement, said method comprising: (a) contacting a sample from saidorganism with a probe nucleic acid comprising an amplifying restrictionendonuclease and a nucleotide sequence complementary to a sequence of atarget nucleic acid containing said genetic or epigenetic element underconditions wherein, if said target nucleic acid is present in saidsample, at least a portion of said target nucleic acid hybridizes to atleast a portion of said probe nucleic acid to form a double-strandedportion of nucleic acid comprising a restriction endonuclease cut site,(b) contacting said double-stranded portion of nucleic acid with arecognition restriction endonuclease having the ability to cut saiddouble-stranded portion of nucleic acid at said restriction endonucleasecut site under conditions wherein said recognition restrictionendonuclease cleaves said double-stranded portion of nucleic acid atsaid restriction endonuclease cut site, thereby separating a portion ofsaid probe nucleic acid comprising said amplifying restrictionendonuclease from at least another portion of said probe nucleic acid,(c) contacting said portion of said probe nucleic acid comprising saidamplifying restriction endonuclease with a reporter nucleic acidcomprising a double-stranded portion of nucleic acid comprising arestriction endonuclease cut site of said amplifying restrictionendonuclease under conditions wherein said amplifying restrictionendonuclease cleaves said reporter nucleic acid at said restrictionendonuclease cut site of said amplifying restriction endonuclease,thereby separating a portion of said reporter nucleic acid from at leastanother portion of said reporter nucleic acid, and (d) determining thepresence or absence of said portion of said reporter nucleic acid,wherein the presence of said portion of said reporter nucleic acidindicates that said sample contains said target nucleic acid, therebyindicating that said organism contains said genetic or epigeneticelement, and wherein the absence of said portion of said reporternucleic acid indicates that said sample does not contain said targetnucleic acid, thereby indicating that said organism does not containsaid genetic or epigenetic element.
 2. The method of claim 1, whereinsaid organism is a human.
 3. The method of claim 1, wherein saidorganism is a mammal.
 4. The method of claim 3, wherein said mammal isselected from the group consisting of bovine, porcine, and equinespecies.
 5. The method of claim 1, wherein said organism is a plant. 6.The method of claim 5, wherein said plant is selected from the groupconsisting of trees, flowers, shrubs, grains, grasses, and legumes. 7.The method of claim 1, wherein said method comprises assessing saidorganism for said genetic element.
 8. The method of claim 7, whereinsaid genetic element is an allelic variant known to exist in the speciesof said organism.
 9. The method of claim 7, wherein said genetic elementis a single nucleotide polymorphism.
 10. The method of claim 1, whereinsaid method comprises assessing said organism for said epigeneticelement.
 11. The method of claim 10, wherein said epigenetic element isa methylated DNA sequence.
 12. The method of claim 1, wherein saidsample is selected from the group consisting of blood samples, hairsamples, skin samples, throat swab samples, cheek swab samples, tissuesamples, cellular samples, and tumor samples.
 13. The method of claim 1,wherein, prior to step (a), said sample was processed to removenon-nucleic acid material from said sample, thereby increasing theconcentration of nucleic acid, if present, within said sample.
 14. Themethod of claim 13, wherein said sample was subjected to a nucleic acidextraction technique.
 15. The method of claim 1, wherein, prior to step(a), said sample was subjected to a nucleic acid amplification techniqueto increase the concentration of one or more nucleic acids, if present,within said sample.
 16. The method of claim 15, wherein said sample wassubjected to a PCR-based technique designed to amplify said targetnucleic acid.
 17. The method of claim 1, wherein said determining stepcomprises determining the amount of said target nucleic acid presentwithin said sample.
 18. A method for an organism for a genetic orepigenetic element, said method comprising: (a) contacting a sample fromsaid mammal with a probe nucleic acid comprising an initial amplifyingrestriction endonuclease and a nucleotide sequence complementary to asequence of a target nucleic acid containing said genetic or epigeneticelement under conditions wherein, if said target nucleic acid is presentin said sample, at least a portion of said target nucleic acidhybridizes to at least a portion of said probe nucleic acid to form adouble-stranded portion of nucleic acid comprising a restrictionendonuclease cut site, (b) contacting said double-stranded portion ofnucleic acid with a recognition restriction endonuclease having theability to cut said double-stranded portion of nucleic acid at saidrestriction endonuclease cut site under conditions wherein saidrecognition restriction endonuclease cleaves said double-strandedportion of nucleic acid at said restriction endonuclease cut site,thereby separating a portion of said probe nucleic acid comprising saidinitial amplifying restriction endonuclease from at least anotherportion of said probe nucleic acid, (c) contacting said portion of saidprobe nucleic acid comprising said initial amplifying restrictionendonuclease with a first nucleic acid comprising a secondary amplifyingrestriction endonuclease and a double-stranded portion of nucleic acidcomprising a restriction endonuclease cut site of said initialamplifying restriction endonuclease under conditions wherein saidinitial amplifying restriction endonuclease cleaves said first nucleicacid at said restriction endonuclease cut site of said initialamplifying restriction endonuclease, thereby separating a portion ofsaid first nucleic acid comprising said secondary amplifying restrictionendonuclease from at least another portion of said first nucleic acid,(d) contacting said portion of said first nucleic acid comprising saidsecondary amplifying restriction endonuclease with a second nucleic acidcomprising said initial amplifying restriction endonuclease and adouble-stranded portion of nucleic acid comprising a restrictionendonuclease cut site of said secondary amplifying restrictionendonuclease under conditions wherein said secondary amplifyingrestriction endonuclease cleaves said second nucleic acid at saidrestriction endonuclease cut site of said secondary amplifyingrestriction endonuclease, thereby separating a portion of said secondnucleic acid comprising said initial amplifying restriction endonucleasefrom at least another portion of said second nucleic acid, (e)contacting said portion of said second nucleic acid comprising saidinitial amplifying restriction endonuclease with a reporter nucleic acidcomprising a double-stranded portion of nucleic acid comprising arestriction endonuclease cut site of said initial amplifying restrictionendonuclease under conditions wherein said initial amplifyingrestriction endonuclease cleaves said reporter nucleic acid at saidrestriction endonuclease cut site of said initial amplifying restrictionendonuclease, thereby separating a portion of said reporter nucleic acidfrom at least another portion of said reporter nucleic acid, and (f)determining the presence or absence of said portion of said reporternucleic acid, wherein the presence of said portion of said reporternucleic acid indicates that said sample contains said target nucleicacid, thereby indicating that said organism contains said genetic orepigenetic element, and wherein the absence of said portion of saidreporter nucleic acid indicates that said sample does not contain saidtarget nucleic acid, thereby indicating that said organism does notcontain said genetic or epigenetic element.
 19. A method for assessingan organism for a genetic or epigenetic element, said method comprising:(a) contacting a sample from said mammal with a probe nucleic acidcomprising an initial amplifying restriction endonuclease and anucleotide sequence complementary to a sequence of a target nucleic acidcontaining said genetic or epigenetic element under conditions wherein,if said target nucleic acid is present in said sample, at least aportion of said target nucleic acid hybridizes to at least a portion ofsaid probe nucleic acid to form a double-stranded portion of nucleicacid comprising a restriction endonuclease cut site, (b) contacting saiddouble-stranded portion of nucleic acid with a recognition restrictionendonuclease having the ability to cut said double-stranded portion ofnucleic acid at said restriction endonuclease cut site under conditionswherein said recognition restriction endonuclease cleaves saiddouble-stranded portion of nucleic acid at said restriction endonucleasecut site, thereby separating a portion of said probe nucleic acidcomprising said initial amplifying restriction endonuclease from atleast another portion of said probe nucleic acid, (c) contacting saidportion of said probe nucleic acid comprising said initial amplifyingrestriction endonuclease with a first reporter nucleic acid comprising asecondary amplifying restriction endonuclease and a double-strandedportion of nucleic acid comprising a restriction endonuclease cut siteof said initial amplifying restriction endonuclease under conditionswherein said initial amplifying restriction endonuclease cleaves saidfirst reporter nucleic acid at said restriction endonuclease cut site ofsaid initial amplifying restriction endonuclease, thereby separating aportion of said first nucleic acid comprising said secondary amplifyingrestriction endonuclease from at least another portion of said firstnucleic acid, (d) contacting said portion of said first reporter nucleicacid comprising said secondary amplifying restriction endonuclease witha second reporter nucleic acid comprising said initial amplifyingrestriction endonuclease and a double-stranded portion of nucleic acidcomprising a restriction endonuclease cut site of said secondaryamplifying restriction endonuclease under conditions wherein saidinitial amplifying restriction endonuclease cleaves said second nucleicacid at said restriction endonuclease cut site of said secondaryamplifying restriction endonuclease, thereby separating a portion ofsaid second nucleic acid comprising said initial amplifying restrictionendonuclease from at least another portion of said second nucleic acid,and (e) determining the presence or absence of said portion of saidfirst reporter nucleic acid, said second reporter nucleic acid, or bothsaid first reporter nucleic acid and said second reporter nucleic acid,wherein said presence indicates that said sample contains said targetnucleic acid, thereby indicating that said organism contains saidgenetic or epigenetic element, and wherein said absence indicates thatsaid sample does not contain said target nucleic acid, therebyindicating that said organism does not contain said genetic orepigenetic element.
 20. A kit for assessing an organism for a genetic orepigenetic element, said kit comprises a probe nucleic acid comprisingan amplifying restriction endonuclease and a nucleotide sequencecomplementary to a sequence of a target nucleic acid containing saidgenetic or epigenetic element, wherein at least a portion of said targetnucleic acid is capable of hybridizing to at least a portion of saidprobe nucleic acid to form a double-stranded portion of nucleic acidcomprising a restriction endonuclease cut site.